Abstract:Objective To observe the changes in blood lipid and inflammatory cells in Wuzhishan (WZS)minipigs and Tibetan minipigs exposed to a high-fat diet environment, and to analyze the expressions of coronary arterydisease-related blood lipid and inflammatory susceptibility genes. Methods Five male WZS minipigs and five Tibetanminipigs were selected for feeding with a high-fat diet for six consecutive months. Blood lipids and blood routine examinationwere performed monthly, and venous blood was taken before and 6 months after high-fat feeding to determine the gene andprotein expression levels of LDLR, LPL, PCSK9, ApoAV, 5-LO, FLAP, MMP-9, ICAM-1 and VCAM-1 in peripheralleukocytes, as well as the levels of CRP and TNF-α. Thoracic aorta samples were taken for pathological examination afterhigh-fat feeding for 6 consecutive months. Results After high-fat diet feeding, the levels of TC, HDL-C, and LDL-C inthe two minipigs were significantly increased ( P <0. 01), and WBC, NEUT% and MONO% in the Tibetan minipigs as wellas MONO% in the WZS minipigs tended to be increased ( P >0. 05). TC and LDL-C levels in the WZS minipigs were higherthan those in the Tibetan minipigs, whereas WBC, NEUT% and MONO% in the Tibetan minipigs were much higher thanthose in the WZS minipigs ( P <0. 05, P <0. 01). After high-fat feeding for 6 months, there were obvious atherosclerotic(AS) plaques in the two minipigs, but the degree of AS lesions in the Tibetan minipigs was more serious. The mRNA andprotein expressions of LDLR and ApoAV in the WZS minipigs were markedly decreased ( P <0. 05, P <0. 01), whereas themRNA expressions of PCSK9 and LPL were markedly increased ( P <0. 05, P <0. 01). The mRNA and protein expressionsof LPL in the Tibetan minipigs were obviously increased ( P <0. 01). Moreover, the mRNA expressions of 5-LO, FLAP,ICAM-1 and VCAM-1, and protein expression of 5-LO in the WZS minipigs were significantly increased ( P <0. 05, P <0. 01). mRNA and protein expressions of FLAP, MMP-9, ICAM-1, and VCAM-1 in the Tibetan minipigs were markedlyincreased ( P <0. 05, P <0. 01). Moreover, mRNA and protein expressions of MMP-9, ICAM-1, and VCAM-1 in Tibetanminipigs were higher than those in the WZS minipigs, whereas mRNA and protein expressions of 5-LO were lower.Conclusions WZS and Tibetan minipigs have various degrees of dyslipidemia and inflammation under a high-fat dietenvironment. Significant heterogeneity exists in CAD-related blood lipids and inflammatory susceptibility genes. Theexpressions of LDLR, PCSK9, and 5-LO in the peripheral leukocytes are more affected in WZS minipigs, while LPL,ICAM-1, VCAM-1, and MMP-9 in the peripheral leukocytes are more affected in Tibetan minipigs.

Abstract:Objective To prepare a Bama minipig model of hereditary tyrosinemia type III, thehydroxyphenylpyruvate dioxygenase (Hpd) gene was chosen to be edited by the CRISPR/ Cas9 technology. Methods Templates (used as in vitro transcripts for Cas9 mRNA and sgRNA) were amplified from pST1374-NLS-flag-linker-Cas9and pGL3-U6-gRNA-PGK-puromycin by PCR, respectively, and subsequently transcribed in vitro into Cas9 mRNA andsgRNA-Hpd. Finally, cas9 mRNA and sgRNA-Hpd were co-injected into the cytoplasm of single cell embryos to generatean Hpd-modified Bama minipig. Results Twenty embryos of Bama minipigs co-injected with cas9 mRNA and sgRNA-Hpdwere transplanted into two pseudopregnant mothers. We obtained four offsprings with a modified Hpd gene. Conclusions Inthis study, we have successfully generated Hpd knockout Bama minipigs with hereditary tyrosinemia type III using the CRISPR/ Cas9 technique, which will be a valuable model for research of the tyrosine metabolic pathway.

Abstract:Objective To perform a genome-wide histone modification profiling of Tibet minipigs and to comparewith the differences in histone modifications in different tissues. Methods Tissue samples of blood, liver, ileum, andmuscle of two three-month-old male Tibet minipigs were collected and used for chromatin immunoprecipitation sequencing.Through quality control, raw data were filtered and compared with the reference genome, then we used MACS software toenrich clean reads in the genome. Results The enrichment positions of different histone modifications differed in the genepromoter region, and the distribution patterns were roughly divided into two types. One type, H3K4me3, was mainlydistributed on both sides near the transcription start site, exhibiting a peak with a narrow range at the downstream position.The second type, H3K9me3, was distributed more evenly throughout the gene promoter, exhibiting a broad peak shape witha low middle and two high sides. Genes affected by the tissue-specific histone modifications differed, and the correspondingfunctional pathways were relatively extensive, but they were all involved in the transcriptional regulation of the RNApolymerase II promoter. Conclusions Our study provides a dataset of histone modification annotations for four types ofdifferent tissues of Tibet minipigs, which will serve as a foundation for future studies using transcriptional regulation of the Tibet minipig as a biomedical animal model.

Abstract:Objective To develop a lentiviral vector containing a Tet-On system for inducible pig urokinase-typeplasminogen activator (puPA) transgene expression under the control of the albumin (Alb) promoter, and to confirm the invitro functionality of the resulting plasmid. Methods First, the fragment of the Alb-enhancer/ promoter was amplified frompAlb-Cre-GH/ BS and subsequently inserted into the XhoI and XbaI sites of pLVX-TetOne by In-Fusion cloning to generatepLVX-Alb-TetOne. Second, the T2A-CopGFP fragment was amplified from pCD823 A-1 and then subcloned into pLVXAlb-TetOne by In-Fusion cloning to construct pLVX-Alb-TetOne-TRE-T2A-CopGFP ( pLATTTG). Finally, the puPAfragment was amplified from H8803 and subsequently inserted into pLATTTG by In-Fusion cloning to produce pLVX-Alb-TetOne-TRE-puPA-T2A-CopGFP ( pLATTPUTG ). To validate the in vitro functionality of the resulting vector(pLATTPUTG), it was transiently transfected into 293T cells, followed by CopGFP assay under an inverted fluorescencemicroscope at 24 h after transfection in the absence of doxycycline (Dox-). Dox was then added into a 6-well plate, and theCopGFP assay was performed at 48 h after the addition of Dox, followed by testing the expression of puPA, CopGFP andFlag by qRT-PCR or western blot. Results The data from enzyme digestion and DNA sequencing demonstrated thatpLATTPUTG was successfully constructed. In the presence of Dox, most cells transfected with pLATTPUTG showed stronggreen fluorescence and a high-level expression of puPA, CopGFP and Flag, whereas in the absence of Dox, no cellstransiently transfected with pLATTPUTG displayed green fluorescence. Conclusions A lentiviral vector containing a Tet-On system for inducible puPA transgene expression under the control of an Alb promoter was successfully generated, which will lay a solid foundation for further study.

Abstract:Objective The shortage of donors is a major problem in organ transplantation, and the generation ofhumanized organs is an effective method to solve this problem. A lack of fumarylacetoacetate hydrolase (FAH) causeshereditary tyrosinemia type I (HTI), which leads to hepatocyte apoptosis and liver damage. Genetic editing of pigs (Susscrofa), establishment of FAH knockout Bama minipigs, combined with human stem cells will facilitate the production ofhumanized stem cell liver. In this study, FAH gene knockout Bama minipigs were generated by CRISPR/ Cas9 based onalpha-1,3-galactosyltransferase (GGTA1) gene knockout pigs,and their health and breeding status were analyzed. Methods Single guide RNA (sgRNAs) were designed to target the porcine FAH gene to construct a pX330 vector that wastransfected into Bama minipig ear fibroblasts. The efficiency of this method was verified. After identification, positive cellswere selected as nuclear donors, and FAH gene knockout Bama minipigs were generated by somatic cell nucleartransplantation technology. The genomic DNA of piglets was extracted and the mutant type was obtained by PCR sequencing. FAH^{+/ -} Bama minipig blood was collected at the age of 5 months, and its blood biochemical and blood routine indexes weretested. Histological changes of livers were analyzed. When FAH^{+/ -} Bama minipigs were sexually mature, they were matedwith wild type (WT) sows, and their breeding ability was evaluated by litter size. Results FAH^{+/ -} and FAH^{-/ -} Bamaminipigs were obtained. Western blotting showed that FAH^{+/ -} Bama minipigs had decreased FAH expression in the liver andkidney. There were no significant differences in the physical and chemical indexes of blood from the FAH^{+/ -} Bama minipigsor WT minipigs. Histological examination revealed vacuolar-like changes in the FAH^{+/ -} pig liver tissues. The litter size ofwild sows bred with FAH^{+/ -} boars was normal. FAH^{+/ -} pigs had normal health status and breeding ability. Conclusions FAH single allele knockout pigs are generated in this study, with normal health and reproductive abilities, which provides a good basis for future studies of the production of double-allele knockout pigs and humanized liver.

Abstract:Objective To establish a nude mouse model of prostate cancer patient-derived xenograft (PDX) andusing this model to evaluate the anti-tumor effects of different treatment regimens. Methods Fresh prostate cancer surgicalspecimens were mixed with Matrigel and transplanted into nude mice supplemented with exogenous androgens. Tumor growthwas continuously monitored, the fidelity was evaluated, and serial passage was performed. Tumor-bearing mice were dividedinto four groups: docetaxel, castration, docetaxel combined with castration and control groups. The tumor volume andmouse body weight were measured during treatment. After treatment, serum total prostate specific antigen (tPSA) level andhistopathological changes of the tumors were detected. Results The PDX model of prostate cancer was successfullyestablished, including hormone-sensitive (D17225) and castration-resistant (C40019) tumors, which retained the mainfeatures of primary tumors. Serum tPSA and other test results showed that docetaxel and docetaxel combined castrationinhibited the D17225 tumor growth significantly. Conclusions The PDX model of prostate cancer is successfullyestablished and can be stably passaged. Docetaxel or combined castration therapy show significant therapeutic effects on the hormone-sensitive (D17225) prostate cancer PDX model.

Abstract:Objective CRISPR/ Cas9 technology was used to construct a taurine transporter gene (solute carrierfamily 6 member 6, Slc6a6) knockout rat, which was used as a stable animal model to study the effects of taurine onneurological diseases. Methods For exon 5 of the Slc6a6 gene, a single-guide RNA (sgRNA) mediated the specificbinding of Cas9 nuclease to the target DNA and cleaved the genomic DNA, which underwent recombinant repair to generatethe gene knockout. Newborn rat genotypes were detected by genotyping and sequencing analysis. The mRNA expression andprotein expression of TauT in rat brain tissues were analyzed by real-time PCR, Western blot and immunohistochemistry.The homozygous Slc6a6 knockout rat was screened out. Results The F3 progeny had 21 Slc6a6 knockout homozygotes(TauT-/ - ), 54 heterozygous (TauT^{+/ -} ) and 27 wild-types (TauT^{+/ +} ). The homozygosity rate was about 20. 59% in the F3generation. The offspring showed a normal Mendelian ratio. No Slc6a6 mRNA was observed in brain tissues from Slc6a6knockout homozygous rats and the expression level of TauT protein was significantly lower than that of littermate-negativerats. Conclusions In this study, the CRISPR/ Cas9 system is used to knockout the exon of the Slc6a6 gene, and the Slc6a6 knockout rat model is successfully constructed.

Abstract:Objective Lumbar spinal needle, indwelling needle, and tracheotomy were used to establish ableomycin-induced pulmonary fibrosis model in C57BL/6J mice,respectively,and to compare the effects of these threemethods on mice. Methods One hundred and twenty C57BL/6J mice were randomly divided into blank, lumbar spinalneedle model, indwelling needle model, and tracheotomy model groups. Mice from different groups were administered byintratracheal instillation of bleomycin using the different methods, respectively. The general condition of the mice (changesin body weight and survival rate) was observed, and the histopathological changes (HE staining and Masson staining) andhydroxyproline level were examined. Results The body weight of the lumbar spinal needle model group and the indwellingneedle model group was significantly higher than that of the tracheotomy model group at 28 days after modeling ( P <0. 01? P <0. 001). There were no significant differences in survival rate among the four groups except between the blank group andthe tracheotomy model group ( P <0. 05). The degrees of alveolitis and pulmonary fibrosis were highest in the tracheotomymodel group, followed by the lumbar spinal needle model group and indwelling needle model group. The degree of alveolitisand pulmonary fibrosis was greatest at 21 days after modeling. The lesions were evenly distributed in the tracheotomy modelgroup, and unevenly distributed in the lumbar spinal needle model and indwelling needle model groups. The lesions in thelung tissue around the trachea were more severe than in other areas in the lumbar spinal needle model and indwelling needlemodel groups. The level of hydroxyproline in the lumbar spinal needle model and tracheotomy model groups was significantlyhigher than that in the blank group ( P <0. 01, P <0. 001). Conclusions In summary, tracheal intubation by lumbar spinal needle is the best method among the three methods for the establishment of a bleomycin-induced pulmonary fibrosis in mice.

Abstract:Objective To examine whether the fasting plasma glucose (FPG) of cynomolgus monkeys (Macacafascicularis) is significantly different between different seasons. The aim of this study was to investigate the correlationbetween FPG and seasonal temperature variation in a year, and to compare the FPG measured by a portable blood glucosemeter with that measured by an automatic biochemical analyzer. Methods Venous blood of monkeys was collected, andblood glucose values of monkeys in different seasons were measured by an automatic biochemical analyzer and portableblood glucose meter. Results FPG levels in cynomolgus monkeys peaked in the winter, reached the lowest level in thesummer, and fluctuated within this range during spring and autumn. Changes in FPG levels during the periods winter→spring and summer→autumn were higher than the periods autumn→winter and spring→summer, with the amplitudechanges of approximately 0. 20 mmol/ L per℃ and 0. 06 mmol/ L per℃, respectively. The differences between FPGmeasured by portable blood glucose meter and automatic biochemical analyzer were in accordance with national standards,indicating that the portable blood glucose meter can be used to detect FPG in cynomolgus monkeys. Conclusions Theresults of this study demonstrate the association of FPG with seasonal temperature variation in cynomolgus monkeys and provide a reasonable analysis of the data in future animal experiments.

Abstract:Objective To establish a mouse model of colon cancer liver metastasis with high metastasis rate,simple operation and reliable outcome, and to serve the studies on prevention and treatment of colon cancer metastasis.Methods Fifteen BALB/ c nude mice were divided into 3 groups of 5 mice each (A, B, and C), and group D consistingof 5 wild type BALB/ c mice. We established four colon cancer metastasis models: spleen planted, spleen preserving, andsplenectomy method using 0. 2 mL of HCT116 or CT26 cell suspension with a cell concentration of 2. 5 × 10⁷/ mL,respectively. We compared the success rates of modeling in the four groups as well as intra-abdominal metastasis, and livermetastasis size and number. Results The success rate of the group A was 100% (5/5). The number of liver metastases inthe group A was low, dispersed, and located in the right lobe of the liver. The mean survival time was (26. 6 + 3. 4) days.The success rate of Group B was 40% (2/5). Metastatic tumors in the group B were scattered on the surface of the liver,and the volume was larger than the group A. The mean survival time was (36. 8 ±4. 2) days. The success rate of the groupC was 100% (5/5). The number of liver metastases in the group C was higher than in the other groups, and multiplemetastatic tumors fused into a mass occupying the right lobe of the liver. The mean survival time was (20. 2 ±2. 6) days.No metastasis was found in the group D. Peritoneal metastasis occurred in the three groups of nude mice (n =2 in Group A,n =3 in Group C) without heart, lung, brain, or kidney metastasis. The histopathological examination of liver metastasesconformed to the characteristics of adenocarcinoma. Conclusions The spleen preserving method shows the highest rate ofmodel establishment, and effectively simulates the route and process of human colon cancer cells travelling to the liver by the blood circulation.

Abstract:Objective To breed and identify Mfge8 gene knock-out (Mfge8^{-/ -} ) C57BL/6 mice, and to examineserological and histological changes in these animals. Methods Genomic DNA was obtained from the tail of young miceand the genotype was determined by PCR and agarose gel electrophoresis. TUNEL assay was used to detect apoptotic cellsin the lung tissues of 12-week-old wild type (WT) and Mfge8^{-/ -} mice. Levels of anti-nuclear antibodies (ANA) and antiendothelialcell antibodies ( AECA) in the serum of 36-week-old WT and Mfge^{-/ -} mice were determined byimmunofluorescence. Results Knockout mice have been successfully generated and maintained that are homozygous forMfge8 gene deletion (Mfge8^{-/ -} ). The number of apoptotic cells in the lung tissues of 12-week-old Mfge8^{-/ -} mice was higherthan in WT mice. In addition, the serum of 36-weeks-old Mfge8^{-/ -} mice, but not WT mice, was positive for ANA andAECA. Conclusions Mfge8 knockout mice with the C57BL/6 genetic background demonstrate a greater susceptibility to autoimmune disease.

Abstract:Objective To investigate the in vivo histological distribution of sialic acid receptors in dogs and itsassociation with the susceptibility to canine influenza virus H3N2. Methods Different organs and tissues were collectedfrom healthy English Springer Spaniel dogs. The type and histologic distribution of SAα2,6Gal and SAα2,3Gal linkage inthese organs were determined using a lectin-based histochemistry method. Organs from dogs artificially infected with H3N2virus were also collected, and were examined by immunohistochemistry to investigate the distribution of virus in vivo.Results SAα2,3Gal and SAα2,6Gal receptors coexisted in most organs and tissues, but also demonstrated differentialexpression in some organs. Among the different organs examined, SAα-2,6Gal receptors generally demonstrated a broaderexpression profile and higher level of expression than SAα-2,3Gal receptors. The in vivo tissue distribution of H3N2 virus inartificially infected dogs was largely consistent with the tissue distribution of sialic receptors, although in a small number oftissues this relationship was not observed. Conclusions Receptors for avian and human influenza virus are widelyexpressed in various tissues and organs of dogs, providing a molecular basis for co-infection of avian and human influenza virus.

Abstract:Objective To evaluate the transplantation effect of erythroid progenitor cells in cloche^{-/ -} mutantzebrafish with congenital primary hematopoietic deficiency. Methods The embryos of zebrafish zTg (gata1:EGFP) werecollected and prepared into a single cell suspension. The gata1⁺ cells carrying green fluorescence were separated by flowcytometry and transplanted into the hearts of 42 hpf cloche^{-/ -} mutant zebrafish by microinjection. The expression of erythroidprogenitor cells in cloche^{-/ -} mutant zebrafish was examined by stereo fluorescence microscopy. Results gata1⁺ cells withgreen fluorescence were successfully selected. The proliferation of gata1⁺ cells with green fluorescence was observed at 2 hafter transplantation, and 16 h of continuous green fluorescence expression was observed. Conclusions Erythroidprogenitor cells have a potential to reconstruct hematopoiesis and provide an experimental basis for the further study of the functional identification of erythroid progenitor cells after transplantation.

Abstract:Objective To investigate the effects of curcumin on glucose transport and phosphatidylinositol 3-kinase (PI3K) / protein kinase B (Akt) signaling pathway in adipocytes of type 2 diabetes mellitus (T2DM) rats. MethodsNinety Sprague-Dawley (SD) rats were randomly divided into normal group, model group (high-fat diet-fed), curcuminlow-dose group (50 mg/ kg), curcumin medium-dose group (150 mg/ kg), and curcumin high-dose group (250 mg/ kg)groups, and rosiglitazone group (1. 35 mg/ kg), each group comprised 15 rats. The body weight of rats was measuredimmediately after administration, 2 weeks after administration and 8 weeks after administration, and blood was collected for12 hours after fasting at the end of 8 weeks, fasting blood glucose (FBG), fasting insulin (FINS), the insulin resistanceindex (HOMA-IR), triglyceride (TG), low/ high-density lipoprotein cholesterol (LDC-C/ HDL-C), and total cholesterol(TC) were measured. The glucose perfusion rate was measured by positive glucose clamp assay, the translocation of glucosetransporter 4 (GluT4) in adipocyte membrane was detected by fluorescence immunoassay,GluT4 in adipocytes was detectedby real-time fluorescence quantitative PCR (qRT-PCR), the expression of GluT4, p-PI3K, insulin receptor substrate 2(Irs2) and p-Akt in intracellular and extracellular membranes of adipocytes were detected by Western blotting. Results Compared with the normal group, the weight of the model group increased immediately and 2 weeks after administration,and decreased 8 weeks after administration; compared with the model group, the body weight of curcumin treatment groupand rosiglitazone treatment group decreased immediately, 2 weeks and 8 weeks after administration ( P < 0. 05). Comparedwith the normal group, the FBG, FINS, HOMA-IR, HDC-L, LDC-L, TC and TG in the model group increased, and theglucose injection rate decreased in a dose-dependent manner; GluT4 mRNA increased in the model group, the proteinexpression of GluT4, Irs2, p-PI3K/ PI3K and pAkt/ Akt decreased significantly; and the differences were statisticallysignificant ( P < 0. 05). Compared with the model group, the levels of FBG, FINS, HOMA-IR, HDC-L, LDC-L, TC and TGin the curcumin treatment group and rosiglitazone group were significantly lower, the rate of glucose injection was significantlyhigher, GluT4 mRNA decreased, the protein expression of GluT4, Irs2, p-PI3K/ PI3K and pAkt/ Akt increased significantly,and the differences were statistically significant ( P < 0. 05), and the difference of curcumin dosage among the three groupswas also statistically significant ( P < 0. 05). Conclusions Curcumin can increase the insulin sensitivity of T2DM rats,which may be achieved by activating PI3K/ Akt signaling pathway to promote the GluT4 membrane translocation in adipocytesand increase the uptake of glucose by adipocytes, thereby alleviating the balance of blood sugar metabolism.

Abstract:Objective To explore the mechanism of the inhibitory effect of microRNA ( miR)-1247 onlipopolysaccharide (LPS)-induced acute pneumonia. Methods Thirty 8-week-old female BABL/ c mice were randomlydivided into three groups: acute pneumonia model group (6 h), acute pneumonia model group (12 h) and normal group,with 10 mice in each group. LPS solution (8 mg/ kg) was instilled into the mouse trachea of the model group, and thenormal group was instilled an equal volume of physiological saline. Autopsy was performed after anesthesia at 6 h and 12 hafter LPS modeling. The blood gas from the celiac artery was measured. The dry and wet weight of lung tissue was measured.qRT-PCR and ELISA were used to detect the expression levels of TNF-α, IL-1β and miR-1247 in the pulmonary tissues.The levels of CCR16, TLR4, IRAK6, TAK, IKK and NF-κB p52 proteins in the lung tissues of each group were detectedby western blot. Results Compared with the normal group, the PaO₂, oxygen index (PaO₂ / FiO₂ ) of the model groupmice was decreased, and the dry-wet weight ratio of the lung was significantly increased ( P < 0.05). However, thedifferences in PaCO₂ and mRNA expression levels of TNF-α and IL-1β between the two groups were not statisticallysignificant ( P > 0.05). The levels of miR-1247 and CCR16 protein expression in the lung tissues of the model group weresignificantly increased at 6 h and 12 h after modeling ( P < 0.05). Compared with the normal group, the expression ofTLR4 on the cell surface was significantly lower ( P < 0.05). Compared with the normal group, the expressions of IRAK6,TAK, IKK and transfected nuclear protein NF-κB in the tissues at 6 h and 12 h after modeling were decreased significantly( P < 0.05).Conclusions The results of this study show that miR-1247 inhibites the increase of various cytokines and proteins in the LPS-induced acute pneumonia model via chemokine ligand CCR16.

Abstract:Objective To compare two method of Mycobacterium tuberculosis culture (the BACTEC MGIT 960rapid culture system and the L?wenstein-Jensen plates (L-J plates) culture method) and to explore the value of theBACTEC MGIT 960 rapid culture system in a Mycobacterium tuberculosis (M.tb)-infected mouse model. Methods Theexperimental groups were divided into BACTEC MGIT 960 and L-J culture groups. Thirty-six female C57BL/6 mice wereinfected with 1. 0×106 colony forming units (CFU) / mL H37Rv via the tail vein. Eighteen mice were treated with rifampicinfor 1 week after infection. Eighteen mice were injected with the same amount of phosphate buffer (PBS) as a control. Thelung, spleen, and liver homogenates of 36 mice were cultured by BACTEC MGIT 960 rapid culture and L-J culture.Results In the BACTEC MGIT 960 culture system group, the time for a positive result in the liver, lung, and spleentissue cultures in the RIF treatment group was (187. 11±10. 20) h, (347. 22±12. 70) h, and (276. 39±13. 09) h,respectively, and in the control group it was (142. 50± 11. 70) h, (251. 67± 16. 63) h, and (230. 28 ± 7. 22) h,respectively. There were significant differences between the two groups ( P < 0. 001). In the L-J plates group, the bacterialload of the liver, lung, and spleen tissue cultures in the RIF treatment group was (5. 15±0. 15) log₁₀ CFU, and (3. 30±0. 23) log₁₀ CFU, and (3. 40±0. 25) log₁₀ CFU, and in the control group it was (5. 90±0. 25) log₁₀ CFU, (3. 88±0. 31)log₁₀ CFU, and (4. 15 ± 0. 30) log₁₀ CFU. There were significant differences between the two groups ( P < 0. 001).Conclusions The culture results are consistent between the BACTEC MGIT 960 and the L-J plates culture methods.However, the time to detect a positive result is reduced by half on average, and the discrimination is higher when using the BACTEC MGIT 960 culture method in an animal model.

Abstract:Objective To explore a simple and easy method for the rapid isdation and culture of corneal stromalcells (CSCs). Methods Two experimental groups A and B were designated. The corneal stromal layer was removed fromtree horny cornea. In the group A, it was cut, digested with 1% type II collagenase for 60 min, and resuspended aftercentrifugation. In the group B, type II collagenase was used first,and then treated with neutral protease digestion. Thegrowth and passage time of primary and passage cells were recorded and compared. Vimentin was detected byimmunohistochemistry and immunofluorescence assays. Results The collagenase digestion method which was used in thegroup A successfully isolated primary cells. After 9 days, the cells were passaged and showed good cell morphology. Thepassage cells grew fast and were passaged for 2 to 3 days. The double enzyme digestion method used in the group B isolatedfewer CSCs by day 10. A small number of scattered cells were observed, which were passaged from 15 to 20 days.Immunohistochemistry and immunofluorescence assays showed positive vimentin expression of the corneal stromal cells inboth groups. Conclusions Both methods can successfully isolated CSCs for culture, but the digestion method with only collagenase is easier and more efficiently to isolat a large number of tree shrew CSCs.

Abstract:Objective To reduce the temperature of waste water and gas through a heat exchanger so it can bedischarged in a polyvinyl chloride (PVC) pipe, and so the heat energy can be controlled by arranging the temperaturesensor at the critical control node. Methods High temperature gas and liquids were exchanged efficiently using a heatexchanger element. The temperature sensor was arranged at the key control node to control the temperature precisely.Results After the sterilizer was stopped, the steam discharge temperature was reduced to 15°C and the water temperatureafter heat transfer was reduced. In the heat recovery process, the heat recovery efficiency reached 72%. Conclusions Theproblem of air pollution caused by high temperature and high pressure gas discharge is reduced allowing energy saving and emission reduction.

Abstract:An increase in the global population and climate warming over the past few decades has increased theglobal epidemic and incidence of Dengue fever, which is now a global public health problem. It is essential to establishideal animal models of Dengue fever to study the infection mechanism and pathogenesis of Dengue virus. In view of thespecies specificity and specific pathogenesis of Dengue virus, research on animal models of Dengue virus infection facesmany challenges. Specifically, immunodeficient mice infected with Dengue virus produce high levels of viral replication andsevere disease manifestations, such as increased vascular permeability and thrombocytopenia; however, some symptomssuch as neurological symptoms differ from those of humans. The humanized mouse model of Dengue virus infection can studythe human immune response to Dengue virus, but it is not comprehensive or accurate. Dengue virus also effectively infectsnon-human primates, although they do not usually develop significant clinical symptoms. Therefore, it is necessary toimprove and establish Dengue virus infection models to support research on the pathogenesis of Dengue virus and thepreclinical evaluation of antiviral drugs and vaccines. This paper brings together the research progress of animal models ofDengue virus infection in recent years, and provides reference information for the research and application of the Dengue virus infection model.

Abstract:Knee osteoarthritis (KOA) is a degenerative and intractable disease with high morbidity. Animal modelsof KOA provide an effective tool for studying the disease pathogenesis, potential treatments and their mechanism of action.A variety of animal models currently exist for studies of KOA, each with its own characteristics and advantages. In additionto the author’s own research on animal models of this disease, this article reviews the literature on method ologies forestablishing animal models of KOA. This article explores the selection criteria and method of preparing experimental KOAanimal models, in order to provide an effective reference for researchers who wish to apply these models to their own work.

Abstract:Ankylosing spondylitis is a chronic inflammatory rheumatic disease which mainly affects the axialskeleton and is closely associated with HLA-B27. As most patients are relative young, the ankylosis and deformity of thejoints caused by ankylosing spondylitis have an important impact on social-economics. Laboratory animal models serve as animportant carrier for the studies of pathogenesis, pathological and pathophysiological mechanisms and intervention ofankylosing spondylitis. In this paper, the research progress in spontaneous, inducible and genetic engineering animalmodels has been reviewed, in order to provide a reference for the basic research, new drug development and clinical treatment of ankylosing spondylitis.

Abstract:Parkinson’s disease (PD) is a neurodegenerative disease common in middle-aged and old people. It isdifficult to diagnose and differentiate PD, Parkinson’ s superimposition syndrome and other motor disorders in the earlystages. Therefore, preclinical research is particularly important, and animal molecular imaging are used. Imaging technologycan dynamically observe the formation of Parkinson’ s disease and the changes of dopamine receptor metabolism andkinetics before and after deep brain stimulation ( DBS) treatment in vivo. It is helpful for the early diagnosis andunderstanding pathogenesis of Parkinson’s disease. At present, small animal PET/ CT,MRI and SPECT are used for thebrain imaging of Parkinson’ s experimental animals for accurate diagnosis. Further research is needed to significantlyimprove the diagnostic efficiency and achieve a high degree of consistency with clinical diagnostic results, and serves thediagnosis and differential diagnosis of Parkinson’s disease and Parkinson’s syndrome. This paper reviews the results ofpreclinical studies on Parkinson’ s disease using molecular imaging technology in recent years, and illustrates thedevelopment trend of imaging medicine, which has a certain reference value for the future diagnosis and treatment of Parkinson’s disease.