Abstract:ObjectiveTo study the early dynamics of lesion formation after vascular injury in LDLR-/- mice by in vivo fluorescence microscopy. MethodsOne hundred and twelve male LDLR-/- mice (18-22 weeks of age) were randomly divided into 14 groups (8 mice in each group). The mice were subjected to bone marrow cell transplantation from GFP⁺/LDLR-/- mice. After 4 weeks, a polyethylene cuff was implanted around the right femoral artery to induce vascular injury. The lesion formation was observed on the injured femoral artery from 1 to 14 days after vascular injury.ResultsAt the 1st day after cuff placement, a markedly large number of GFP positive cells were clearly observed in high-speed blood flow. At the 3rd day after the vascular injury, GFP positive cells accumulated in the inner vascular wall and formed punctate lesions. At the 6th day after the vascular injury, GFP positive cells in the inner vascular wall formed irregular flakes, and a small amount of the adventitia tissue proliferated, corresponding to the inner lesion area. At the 9th day after the vascular injury, the adventitia tissue was obviously proliferated and a large number of GFP positive cells embeded. The vasa vasorum were clearly seen running in the adventitial layer. At the 14th day after the vascular injury, the adventitia tissue proliferated considerably with largely GFP positive cells and could not be observed in the lesion through adventitia. After stripping the outer tissue of vessel wall, the fluorescent lesion formed by GFP positive cells was clearly lining in the intima area.ConclusionsThe results of our study demonstrate that the vascular remodeling lesion in the early stage in LDLR-/- mice has a trend of “inside out”. The dynamics of lesions has a close correlation with bone marrow-derived cells that circulating in the blood flow. There is distinct influence of the vascular lesions on extravascular fibrosis. 

Abstract:ObjectiveIn order to reveal the molecular mechanism of the albino mutant Cricetulus barabensis, the tyrosinase gene of Cricetulus barabensis and the albino mutant Cricetulus barabensis was cloned, sequenced, and comparatively analyzed. MethodsThe Tyrosinase cDNA was amplified from the total RNA of the skin of Cricetulus barabensis and its albino mutant Cricetulus barabensis by reverse transcriptase (RT-PCR). According to the conservative region of tyrosinase gene of mice and rats, three pairs of primers were designed to link three fragments to a long epitope by overlap extension. Then the three fragments were amplified and sequenced and finally spliced into the complete sequence. The mutation site was identified through DNA matching software.ResultsThe tyrosinase genes of Cricetulus barabensis and the albino mutant Cricetulus barabensis were cloned and the mutation site was measured by DNA sequence analysis software. The results showed that they have the same coding region and there is no mutation.ConclusionsThe reason of the albino trait of the albino mutant Cricetulus barabensis is different from that of albino trait known in mice, not by the mutation of tyrosinase gene coding region, and reqires further investigation.

Abstract:ObjectiveTry to analyze the gonadotropin-releasing hormone (GnRH) post-receptor signal transduction mechanism of the expression of LHβ mRNA in Rats.MethodsThe gonadotropic hormone (GTH) cells of rat adenohypophysis was stimulated by GnRH impulse at high frequency after cAMP in them was excited by forskolin (FSK) or inhibited by SQ 22536. The Ct value of LHβ mRNA was detected by real-time quantitative polymerase chain reaction, and was compared with that of the blank control group.ResultsThe Ct value of LHβ mRNA was significantly falling with the rise of cAMP in GTH cells and significantly rising with the falling of cAMP.ConclusionsThe cells stimulated by GnRH impulse at high frequency respond to LH βmRNA and cAMP is the post-receptor signal transduction pathway. 

Abstract:ObjectiveTo study the tumor growth and pulmonary metastasis in mice transplanted with different number of luciferase-labeled mouse breast cancer cells, and to provide important information for drug screening and cancer mechanism research. MethodsThe vector containing luciferase gene was constructed and transfected into mouse breast tumor cell line 4T1 cells and selected with G418 to obtain stable Luc-expressing clones. The cells in logarithmic phase was collected and diluted to 1×10⁸,5×10⁷,2×10⁷ and 1×10⁷ cells/mL. Then 0.1 mL cell suspension was inoculated into the second mammary fat pad on the right side of normal BALB/c mice to establish the tumor models. Their tumorigenesis and metastasis were analyzed in vivo.ResultsThe stable Luc-expressing cell lines were obtained which had no obvious difference with parental cells in tumorigenicity. The whole-body optical imaging found that tumor could be formed after the cells were inoculated orthotopically. After 28 days, the tumor sizes were similar among the four concentrations, while there were two mice each died at 5×10⁶ and 1×10⁷ cells concentrations. After 31 days, the tumor metastasis showed different degree among the four concentrations. The metastasis became more seriously with time passed. However, after 42 days, deaths of mice occurred in succession.ConclusionsThe number of 1×10⁶ cells is the best concentration, not only induces obvious pulmonary metastasis but also shows a survival time longer than 45 days, compared with that induced at other higher concentrations. 

Abstract:ObjectiveTo improve the intracellular cytokines staining (ICS) assay and serve the detection of SIV-specific cellular immunity in SIV-infected rhesus monkeys.MethodsThree polyclonal activators were used for PBMC stimulation, and the best positive stimulators and the reasonable stimulating time were tested and determined. Five concentrations of SIVmac239 mixed peptides pool were designed to stimulate PBMC from SIV-infected rhesus monkeys, followed by culturing in vitro and detected with flow cytometry at different times, and the optimal concentration of SIV mixed peptides pool and the best stimulating time were ascertained finally. Then, this method was used for detection of the SIV-specific cellular immunity in the monkeys. ResultsCombination of PMA+ ionomycin could be used as the best positive stimulator. Cytokines secreted from T cells were detected at the highest level by 2 μg/mL SIVmac239 mixed peptides pool at 37℃ and in 5% CO₂ culturing for 16 h in vitro. ConclusionsOptimization of the experimental conditions of ICS has been successfully accomplished, and it may be useful for pre-clinical evaluation of AIDS drugs and vaccine research.

Abstract:ObjectiveTo screen markers used in genetic quality detection of inbred strain HAN of Gobiocypris rarus. MethodsLiving donor tranplatation of scales and polyacrylamide gel electrophoresis of isozyme were used for the genetic quality control of HAN inbred strain.ResultsThe survival rate of allotransplanted scales in the inbred strain HAN was significantly higher (80%) than that of 7.4% in the wild population. Six isozymes presumably were encoded by 13 gene loci, 11 of which were found monomorphic in the wild population, and 2 polymorphic loci (est2, est3) with a mean proportion of 15.56%. However, no polymorphic loci was found in F22. In addition, no difference between individuals was found in the inbred stock. ConclusionsAfter successive mating brother and sisiter, inbred stock of G. rarus has reached a high degree of genetic purity. It is feasible to perform genetic quality control of HAN inbred strain of Gobiocypris rarus by scale allotransplantation and gel electrophoresis on Est and Mp. 

Abstract:ObjectiveTo establish a mouse model of tuberculosis by simulating natural infection and make a comprehensive evaluation to the pathological changes of this model.MethodsC57BL/6J mice were infected with Mycobacterium tuberculosis H37Rv by aerosol inhalation. At 4^th, 6^th and 8^th week after infection, the infected mice were scanned by micro-CT and the lungs and spleens were taken for pathological examination (HE and acid-fast staining) and colony counting.ResultsThe deterioration of lung infection in mice were indicated by visual observation and micro-CT scanning results, and diffuse infection throughout the whole lungs at 8^th week. The presence of granulomatous consolidation and Mycobacterium tuberculosis in the lungs were revealed by HE and acid-fast staining. ConclusionsThe mouse model of tuberculosis has been successfully established according to the comprehensive analysis by gross inspection, histopathology, imaging and colony counting. Although this mouse model could not completely represent the same situation of tuberculosis in humans, it may find reasonable application and promote researches relevant to tuberculosis.

Abstract:ObjectiveKcna3 is one of mitochondrial K⁺ channels, which may be associated with apoptosis of myocardium during heart ischemia/reperfusion injury. The aim of this study was to establish a Kcna3 gene knockout mouse model to study the impact of mitochondrial potassium channel in heart ischemia/reperfusion injury, and explore its relationship with heart failure. MethodsA homologous recombination vector was constructed with BAC vector. Strain 129 mouse embryonic stem (ES) cells were targeted knockout of Kcna3 by the homologous recombination vector, and screened with G418 plus GANC. The Kcna3-knockout embryonic stem cells were microinjected into blastula of C57BL/6J mice after superovulation. F1 hybrid mice were bred to obtain mouse aggregation chimeras, and were identified by PCR plus sequencing of tail genomic DNA. ResultsEight Kcna3+/-mice were harvested and identified from 40 gray mice. ConclusionsThe heterozygous Kcna3-knockout mouse model is successfully established and it lays a foundation for further breed homozygous mice. It is a good model for study of the relationship between mitochondrial k⁺ channel abnormality with heart failure, and is of importance in research of drug screening.

Abstract:ObjectiveTo study the effects of estradiol-17 beta (E₂), nonylphenol (NP), polychlorinated biphenyls (PCBs), Cd²⁺, Zn²⁺ and their mixture on the SOD activity in Tanichthys albonubes. MethodsT. albonubes were exposed to the substances mentioned above at different concentrations for 14 days. The activities of SOD at 7 days and 14 days were detected. ResultsThe activity of SOD was significantly raised after the treatment at lower E₂ levels for 7 days. It did not change obviously when the concentration of E₂ was more increased or the exposure time was protracted. At the low concentration of NP, the activity of SOD almost had no changes, but it was raised observably at high concentration. The activity of SOD was decreased after exposure to middle and high PCB concentrations for 7 or 14 days, and so did it in low PCB concentrations for 14 days. Furthermore, the higher the concentration of PCB or the longer the exposure time, the more the SOD activity was decreased. All the Cd²⁺ and Zn²⁺ concentration groups designed in the study were found to restrain the SOD activity, and the SOD activity decreased more significantly with the extension of exposure time. ConclusionsThese substances obviously impact the SOD activity in T. albonubes. A good concentration-response relationship is found between SOD activity and E₂, NP, PCBs, Cd²⁺, Zn²⁺exposure concentrations. Compared with individual effects, the joint effects of these compounds are related to exposure time and (or) the mixture concentrations. Most of the combinations show an enhanced toxicity.

Abstract:ObjectiveTo investigate the effects of indigowoad root polysaccharide (IRP) at different doses on the expression of IgG and SIgA positive cells in duodenum of colostrum-deprived Sprague-Dawley (SD) filial rats. MethodsForty breeds of the filial rats at day 0 were randomly divided into groups A-E:(A) colostrum group, (B)milk group, (C) milk and the high doses of IRP group, (D) milk and the medium doses of IRP group, and (E) milk and the low doses of IRP group. The groups A-E of filial rats were administered 0.9% saline, 0.9% saline, low, medium and high doses of IRP, respectively. Six of them were randomly sacrifieced to collect materials at day 0,7, 4,1, 28, respectively. The distribution and changes of IgG- and SIgA-positive cells and their secretions in the duodenum of the filial rats were examined by immunohistochemistry and using a Moticam 2306 image analysis system. ResultsIgG- and SIgA-positive cells were firstly distributed in the intestinal luminal lamina propria, and then they were also located in the villus lamina propria, and among epithelial cells and in the muscular layer by days. The numbers of IgG- and SIgA-positive cells were increased by days, and the quantities of them in the group C, D and E were larger than that in the group B between d7 to d28. The groups E, D and C had the most immunological enhancement effect on days 7-14, days 14-21, and days 21-28. ConclusionsIRP can enhance the expression of IgG and SIgA in duodenal cells of colostrum-deprived filial rats. The optimal dosage of IRP can be reduced by days. 

Abstract:ObjectiveTo investigate the differences between immune-related pain induced by antigen-specific immune complex and inflammatory pain induced by formalin. MethodsFifteen adult health SD rats were randomly divided into control group, formalin group and immune-complex group with 5 rats in each group. The right hindpaws of rats were respectively injected with PBS, formalin and rat IgG immune-complex. The changes of hindpaw thickness and pain behavior were observed at 0,0 min, 1 h, 2 h, 4 h, 8 h and 12 h after injection. Serum macrophage migration-inhibitory factor (MIF) was detected by ELISA. Expression of MIF in the hindpaw skin and spinal cord was determined by RT- PCR. Results1. Changes of nociceptive behavior:rats in the formalin group showed significant nociceptive behavior immediately, such as licking foot, limping and highly swollen foot which could not touch the ground. Abnormal nociceptive behavior was lasting more than 1 hour after injection and alleviated after then. Pain threshold of rats in the immune complex group obviously decreased at 4 hours after injection without red swollen hindpaw. Expression of MIF of skin and spinal cord in the rats of formalin group were significantly higher than that in the control group (P<0.05). There was statistically not significant difference of MIF expression between the immune complex group and control group. ConclusionsMacrophage migration inhibitory factor contributes to the pathogenesis of inflammatory pain. But no evidence shows that MIF also anticipates in the pathogenesis of immune-related pain. There are some differences in the mechanisms of the immune-related pain and inflammatory pain induced by formalin.

Abstract:Objective To observe the typical ECG appearance of the inbred MIJ and HFJ rats and compare with that of Wistar rats. MethodsSix of each gender and strains of rats were selected as one group. The rats were anesthetized and fixed on a board. A needle electrode pierced into the skin about 2—3 mm. FX-102B ECG device was applied to perform the ECG record. ResultsAs same as that of Wistar rats, both the male and female MIJ and HFJ rats showed a sinus rhythm, and the heart rhythm was regular. No marked differences were showed between the genders and the MIJ and HFJ rats. However, compared with the male Wistar rats, the heart rate of male HFJ and MIJ rats was higher. The electrical axis of heart in HFJ and MIJ rats was as the same as that of the Wistar rats, mostly ranging from 0°—90°. All characteristics of P-waves and QRS waves in the HFJ and MIJ rats were the same as that of Wistar rats. But there were apparent differences in the amplitude and periods of different waves between the strains and genders. ConclusionThere are characteristic ECG manifestations in inbred MIJ and HFJ rats.

Abstract:Objective To establish a rabbit model of adnexal torsion, with typical lesions, good stability, and simple operation, and to explore pathological changes and the role of iNOS in the ovary after preserved surgery. MethodsForty Japanese long-ear white rabbits were randomly divided into adnexal torsion (AT) model groups (n = 32) and control group (n = 8) using a random digit table. In the model groups, the left adnexes of rabbits were twisted clockwise 1080° degrees. After that, a 4/0 silk thread was put across the 3 spiral rings of the rotated adnex and sutured to fix it on the left abdominal wall. Then the rabbits in model groups were divided into 4 groups (8 rabbits in each group). Both ovaries of each rabbit were removed at 4,8, 2,6 h after the adnexal torsion, respectively. In the control group, the ovaries of the rabbits were removed at 96 h after the sham operation. All the right ovaries were removed and served as internal control. The left ovaries were taken a half for pathological examination, and the rest for biochemical detection of iNOS. ResultsAt 24 h after the generation of adnexal torsion and its fixisation on the left abdominal wall, ovarian congestion, infiltration of inflammatory cells and edema were seen. More prominent infiltration of inflammatory cells and disorders of cell structure were found after 48 h. After 72 h, a lot of infiltration of inflammatory cells and focal necrosis occurred. Diffuse necosis of cells was observed after 96 h. Pathological examination of the ovarian tissue revealed the same phasic changes. Biochemical detection of the iNOS levels in each group (left vs. right) were as follows:24 h:(3.542±0.987) vs. (1.521±0.214)U/mgprot, P<0.01; 48 h:(4.986±1.321) vs. (1.832±0.321) U/mgprot, P<0.01; 72 h:(7.991±1.832) vs. (1.124±0.357)U/mgprot, P<0.01; 96 h:(6.991±1.227) vs. (1.732±0.572)U/mgprot, P<0.01. In the AT groups, the iNOS expression in the ovaries reached a peak at 72 h and declined at 96 h after the modeling of ovarian torsion. ConclusionsA rabbit adnexal torsion model has been successfully established. It has favorable features such as simple operation, typical lesions and good reproducibility, and this model well simulates the pathophysiological process of adnexal torsion in women. Threrefore, it may have great significance in further study of adnexal torsion. It is preliminarily concluded that the ovarian injuries become irreversible at 72 h after adnexal torsion, and it is the critical time point of clinical treatment of adnexal torsion with preservation of the ovaries.

Abstract:Objective To explore the correlation of liver receptor homolog-1(Lrh-1; NR5A2) gene nucleotide sequence and ovulation number in mice. MethodsFive primers were designed according to the sequence of NM_001159769 and amplified the coding sequence of Lrh-1 gene. Single-strand conformation polymorphism was used to analyze the correlation of difference in Lrh-1 nucleotide sequence and ovulation number. Results(1) There were polymorphisms in P2 and P5 PCR products. The P2 PCR products had two genotypes:AA and AB. The AB genotype had one base mutation of C674A, and the mutation led to amino acid change of Q140K. (2) The P5 PCR products had three SSCP-types:SSCP-1, SSCP-2 and SSCP-3. The nucleotide sequences of SSCP-1 and SSCP-3 had 25 base deletion than SSCP-2 type. Five base mutations between SSCP-3 and SSCP-2,4 base mutations between SSCP-3 and SSCP-1 were found. When BLAST in NCBI, the nucleotide sequence of SSCP-2 was like NM_001159769, but SSCP-1 sequence was like NG_012313.1. The base mutation of A1652G caused alanine changed to threonine and the base mutation of G1678C caused tyrosine changed to termination codon in peptide chain of SSCP-3 type. (3)There was no significant difference of average ovulation numbers between AA genotype (27.27±8.52) and AB genotype (25.92±11.73) in P2 PCR products (P>0.05). In P5 PCR products, the SSCP-2 type (35.00±14.58) had a significantly higher average ovulation number than SSCP-3 type’s (19.50±7.94) (P<0.05). ConclusionsDefinite correlations of Lrh-1 gene and ovulation number have been established in mice. The results will contribute to the research of Lrh-1 gene molecular mechanism and regulatory pathway in animal reproduction.

Abstract:ObjectiveTo obtain the nucleotide sequence of the complete mitochondrial DNA (mtDNA) of Cricetulus griseus to provide molecular data for mitochondrial disease models. MethodsThe complete mitochondrial genome of Cricetulus griseus was sequenced with 27 primers and their PCR amplications, the genomic structural characteristics and gene mapping were analyzed, and to discuss the phylogenetic relationship among 5 rodent species whose complete mitochondrial genome sequences are available in the published databases. ResultsThe complete genome contains 16 283 base pairs and encodes 37 genes,including 13 protein-coding,2 ribosomal RNA and 22 transfer RNA genes. The base composition for the four nucleotides is 33.53 % A, 30.50% T, 12.98 %G and 22.80% C. ConclusionsThe genome of Cricetulus griseus is very similar to that of other vertebrate mitochondrial genomes. Some of the protein-coding regions and the tRNA genes are highly homologous to those of other rodents. The evolution tree built from the homology of 5 kinds of laboratory animals is consistent with the traditional taxonomic results.

Abstract:ObjectiveTo evaluate the formation and mechanism of non-alcoholic fat liver disease (NAFLD) in three species of rodents by analyzing effect of species differences on replication of NAFLD models. MethodsTwenty SD rats, 20 ICR mice and 20 Mongolian gerbils were assigned randomly into 2 groups respectively:model group (fed with high fat diet) and control group (fed with normal diet). After 16 weeks, hepatic pathology was observed with HE and Mallory''s trichrome staining, and serum lipid levels (CHO, TG, LDL-c, HDL-c) and liver function (GOP, GPT), liver index, antioxidant enzyme activity (SOD, GSH-PX, CAT), hydroxyproline, free fatty acid, MDA of liver were detected. ResultsCompared with the control group, Hyp, CHO, TG, LDL-c, HDL-c, liver index, GOP, GPT, MDA, FFA were significantly increased, whereas SOD, GSH-PX, CAT were significantly decreased in Mongolian gerbils (P<0.05, P<0.01); pathology showed hepatic fibrosis; CHO, liver index, GOP, GPT, FFA, SOD were significantly increased, whereas MDA, GSH-PX, CAT were significantly decreased in SD rats (P<0.05, P<0.01), and formed focal non-alcoholic steatohepatitis; CHO, LDL-c, HDL-c, liver index, CAT were significantly increased, whereas MDA was significantly decreased in ICR mice (P<0.05, P<0.01), and the liver histology was normal. ConclusionsThere are obviously statistical differences in lipid metabolism, liver function and oxidative stress among the three species of rodents. They form different NAFLD models:Mongolian gerbils form liver fibrosis model with high TG, CHO, SD; rats form focal fatty hepatitis with high CHO; and ICR mice form hypercholesterolemia model without pathological changes in the liver.

Abstract:ObjectiveTo clone the full length cDNA of Uncv gene in mice and to express the gene in eukaryotic cells.MethodsRT-PCR assay was applied to clone the full length coding region of the Uncv gene and constructed its expression plasmid pcDNA 3.1-Flag/Uncv. The recombinant plasmid was transfected into HeLa cells and the fusion protein was identified by Western blot analysis.ResultsThe complete coding sequence was obtained and cloned into the pcDNA 3.1-Flag vector. The recombinant pcDNA 3.1-Flag/Uncv plasmid was transiently expressed in HeLa cells. HeLa cell clones expressing fusion protein with molecular weight of about 95×10³ were obtained. ConclusionsA recombinant eukaryotic expression plasmid of Uncv has been successfully constructed and expressed in HeLa cells. It may provide a foundation for further biological studies of Uncv gene.

Abstract:ObjectiveTo purify IgG from hamster serum, and to prepare the rabbit anti-hamster IgG-HRP, to be used for detection of the antibody from Sendai virus (SV)-infected hamsters. MethodsUsing HiTrap Protein-A affinity chromatography column to purify the IgG from hamster serum. The purity of the hamster-IgG was identified by SDS-PAGE analysis. To produce the hamster against rabbit IgG, and to measure the titer of rabbit anti-hamster IgG by double immunodiffusion. To purify the rabbit anti-hamster IgG by affinity chromatography and prepare the rabbit anti-hamster HRP-conjugated antibodies. The working concentration of the rabbit anti-hamster IgG-HRP was detected by direct ELISA and Western blot, and detect the antibody from Sendai virus-infected hamster by enzyme immunoassay (IEA). ResultsThe hamster IgG was purified up to 95% determined by SDS-PAGE analysis, and the titer of the antisrum was up to 1:64 assessed by double immunodifusion. The working concentration of the rabbit anti-hamster IgG was up to 1:5000 measured by direct ELISA, and 1:2000 measured by Western blot. The IEA titer was 1:2000. ConclusionsThe method has been established for efficient purification of IgG fraction from hamster serum, and the polyclonal antibody of the hamster-IgG and the rabbit anti-hamster IgG-HRP are prepared, which may provide a basis for development of serological test system of microbial pathogens of hamster.

Abstract:ObjectiveThe expression of Smad3 protein in the pulmonary fibrosis induced by paraquat (PQ) was detected by immunohistochemistry. Methods57BL/6J male mice(n = 58)were divided randomly into the experimental group and control group. Pulmonary fibrosis was induced by intraperitoneal injection of PQ(10 mg/kg)in the experimental group(n = 48), while physiological saline was used in the control group(n = 10)by the same method. The mice were sacrificed on days 2,5, 7 and 14 in the experimental group and the control ones on day 7. The lungs were taken for histological examinatioon using HE staining and the expression of Smad3 protein was determined by immunohistochemistry. ResultsThe histological changes including edema, hemorrhage and inflammatory cell infiltration were seen in the lung tissues of mice after PQ administration on days 2 and 5. There were slight collagen deposition and plaque-like fibrosis on day 7 and apparent fibrosis on day 14. The Smad3-positive signs appeared in the nucli of infiltrating macrophages and many type Ⅱ alveolar epithelial cells on days 2,5 and 7. The positive expression of Smad3 proteins was positively correlated with the amount of infiltrating macrophages. On day 14 the faint Smad3-positive expression was detected in the nuclei of fibroblasts in hyperplastic foci. ConclusionsThe expression of Smad3 protein is abnormal during pulmonary fibrosis induced by PQ and Smad3 plays an important role in the development of pulmonary fibrosis.