Table of Content

25 March 2004, Volume 24 Issue 1

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A Comparison Between the Application of Flexible Film Isolators and Individual Ventilated Cages

G C Bantin

2004, 24(1):  1-6. 

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Establishment of Hepatic Oval Cell Proliferation Model and Purification of Hepatic Oval Cell

QIN Ai-lan1,2, ZHOU Xia-qiu1, XIE Qing1, YU Hong1，ZHANG Wei1

2004, 24(1):  7-10. 

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To establish a rat model for hepatic oval cells proliferation and to analyze the characterization of oval cell,SD rats （about 150 g） were given 2-acetylaminofluorene （2-AAF）6 days before partial hepatectomy and 7 days after it. Liver tissue specimens were obtained every 2?3 days after partial hepatectomy and were investigated by HE staining and immunohistochemical staining for Brdu，OV6，cytokeratin-19，albumin，AFP, connexin-43 and C-kit. Oval cells were enriched by magnetic activated cell sorting （MACS） using C-kit antibody.RT-PCR was per-formed on sorted cells for the albumin，AFP and CK19 messenger RNA expression. Results：Ob-vious oval cell proliferation was seen in the liver in 10 mg/kg，15 mg/kg,20 mg/kg groups and o-val cell Brdu incorporation test is positive. Oval cells expressed the specific marker OV6，hepatic cell marker albumin, biliary cell marker CK19，fetal liver cell marker AFP and connexin-43 as well as hematopoietic stem cell receptor C-kit. More than 90% sorted oval cells were OV6 positive and all expressed the albumin,AFP and CK19 gene. Conclusions：Rat model for hepatic oval cell proliferation can be established with 2-AAF/partial hepatectomy at dosage of 10?20 mg/kg body weight. And such oval cells were hepatic stem/progenitor cells which expressed OV6/ CKl9/albumin/AFP/connexin43/C-kit. Oval cell population can be highly enriched with MACS using C-kit.

Establishment of Three Models on Chondrocyte Apoptosis

ZHANG Mei1，LI Ping1, WANG Jian1, ZHAI Zhi-ming1, HU Wen1, CHEN Zhao-hui2

2004, 24(1):  11-15. 

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To investingate the effect of drug on apoptosis of chondrocyte in osteoarthritis, the three chondrocyte apoptosis models were established，treated with sodium nitroprusside （SNP）、all-trans retinoic acid （ATRA）and tumor necrosis factor-a （TNF-a） respectively. The apoptosis rate were analysed by Annexin V/PI with flow cytometry and the characteristic alterations of morphology were detected by electron miscroscope. When rabbit chondrocyte were incubated with SNP （2mmol/l），and ATRA （10^-4 mmol/l），and chondrocyte coming from fetal were incubated with TNF-a（30/μg/L） for 24h,they all could induce apoptosis in chondrocyte in vitro. The re-sults suggested that the chondrocyte apoptosis models could be established by sodium nitroprus-side、all-trans retinoic acid and tumor necrosis factor-α respectively.

Application of Sperm Cryopreservation and IVF on KM Mouse Breeding

LU Ye1,2, TANG Yi-min1, LIU Li-jun, Mao Jia-xia1,Bao Shi-min1，Fu Jie1, XIE En1, XUE Zhi-mou2，XU Ping1

2004, 24(1):  16-18. 

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Frozen/thawed sperm,obtained from KM mice in Wu Han Institute of Biologic Products and Shanghai Biochemistry Pharmaceuticals Factory, were fertilized in vitro with superovulated oocytes from Shanghai Laboratory Animal Center KM mice. The 2-cell embryos were transplanted to SPF pseudopregnant ICR recipients. Frozen/thawed sperm from KM mice in Shanghai Laboratory Animal Center were used as control. This study showed that there were no significant difference in the thawed sperm activity rate, IVF rate and offspring produced rate among the three colonies and the results of microbiological monitoring revealed that all pups were SPF. Compared with traditional breeding method, the sperm cryopreservation and IVF technique are more efficient and convenient in standardization breeding of KM mouse.

Seeding of ePTFE Prostheses Interposition Grafts with Bone Marrow-derived Endothelial Cells in Dogs

WANG Yi1，CHEN Yi-ren1，DAI Kun-yang2, Niu Hong-wen2, Wu Bo2, LI Li2

2004, 24(1):  19-22. 

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To evaluate the value of circulating endothelial cells as the resource of endothelial-ization,by seeding of ePTFE prostheses interposition grafts in dogs, making prepare for endothe-lialization of ePTFE in vivo. Methods：Dog bone marrow monocytes were isolated by centrifugation. The procedure of endothelial lining ePTFE prostheses（4 mm×4 cm and 8 mm×5 cm）were finished and interposed in carotid artery and inferior vena cave in 20 dogs. Doppler ultrasound were performed before animal were killed 2 weeks and 2 months after surgery, and graft segments were grossly evaluated and then under pathological examination, immunocytochemistry ob-servation and scanning electron microscopic analysis. Results：Patency rates for seeded grafts in carotid artery group were 100% （5/5）and 60% （3/5）at 2 week and 2 month respectively, control grafts had a patency rate of 40%（2/5）and 0%（0/5）at corresponding times；Patency rates for seeded grafts in inferior vena cava group were 83. 3%（5/6）and 50% （2/4）at 2 month in experiment grafts and control grafts respectively.Scanning electron microscopy showed endothelial cells coverage rates were different between experiment and control groups at carotid grafts （2 month）（84%±11% vs 43%±15%，P＜0.05），so did at inferior vena cava（95%±6% vs 77%士 13%，P＜0. 05）. Neointimal thickness was thinner in experiment grafts as compared with those of control grafts at 2 month（116±28 μm vs 162±30 μm，P＜0.05； 91±24 vs 143士35 pm， P＜0.05）. Conclusion：Lining ePTFE protheses with bone marrow-derived endothelial cells fa-cililated luminal endothelialization and induced less chronic intimal proliferation；As the potential source，circulating endothelial cell had definite value in the endothelialization of protheses in vivo.

Changes of Immune Function in Closed Colony of KM Mice after Biocleaning

LU Xiao-cong1，LIN Qiao2, WANG Yu-E2，WANG You-Wei1

2004, 24(1):  23-25. 

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In order to observe the changes of immune function in KM mice before and after biocleaning,immunological examinations were conducted. The function of antibody formation cells was 557.39±85.7 and 368. 62±65.23，the antibody level in serum was 67.8±26.08 and 46.4± 15.45，the function of removing carbonaceous granule index K was 5.24 ± 0.96 and 4.61±0.61，a was 0.0032±0.0023 and 0.0025±0.001，the phagocytic function of macrophages in mice’s abdominal cavities 0.41±0.66 and 0.36±0.06，the mice’s lymphocyte transformation induced by ConA （OD value） 0.023±0.02 and 0.031±0.001 ,the mice’s DTH induced by DNFB 10.85±4.34 and 16.29±4.21 before and after biocleaning respectively. It was suggested that some of examined immune function changed significantly after bioclcaning such as the phagocytic activity, B lymphatic cells activity and antibody levels. However, no changes were observed in mice’s lymphocyte transformation induced by ConA.

Metabolism Changes of Experimental Avascular Necrosis and Repair of Dogs Femoral Head

WU Shi-liang1, XU Lan1，ZHOU Yin-hui1, SHI Ning1，WANG Jin-Xi2

2004, 24(1):  26-29. 

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To observe the metabolism changes in the course of repair and avascular necrosis of the femoral head, the experiment was carried out on 26 adult dogs,6 of them were used as control. Avascular necrosis of the left femoral head was induced by freezing method with liquid nitrogen, and the right half being used as self-control. The 20 dogs were divided into four groups, 5 dogs in each which was sacrificed in 15、60、120 and 180 days after operation respectively. All the femoral heads were examined by biochemical methods. The cross-link and non-cross-link collagen contents, aminohexose contents of each cancellous bone and cartilage' bone of fresh femoral-heads，bone tissue collagenase activity and calcium contents were detected. The collagen contents of cancellous bone, including cross-link and non-cross-link which had no significant change in 15 day, reduced in 60 day group and further reduced in 120 or 180 day groups,Activity of tissue collagenase of cancellous bone reduced in 15 day after experimental necrosis （P＜0.01）， but significant increased in 60 day group （P＜0.01），and in 120 or 180 day groups （P＜0.05）. The calcium level had a same change as in the cancellous bone. The results suggested that the collagen and collagenase may play an important role in the course of avascular necrosis and repair of femoral head.

Establishment and Identification of Minipig Model with Myocardial Infarction and ischemia

XU Chang-de1，XU Lan-wen2,YAO Rui-ming3,ZHANG Qing-yong3, SHIHong-cheng1, CHEN Shao-liang1

2004, 24(1):  30-33. 

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To establish the minipig model with the myocardial infarction or ischimia,and observation marker proving the model. 12 minipigs with body weight 16-22 kilogram were divided into two groups，one was treated with circumflex branch ligation for the myocardial infarction,the other was treated circumflex branch with ameroid constrictor for myocardial ischemia. Both the models underwent exercise and resting myocardial perfusion imaging with single photon emission computed tomography （SPECT） using ^99mTc-MIBI,they also underwent the staining with 2，3-triphenyltetretrazolium chloride in order to distinguish the myocardial infarction or ischemia with isolated heart. The results showed that the left ventricle of the model was observed the defect of ^99mTc-MIBI in the infarction region where the staining of TTC was no uptake. The other model was observed rarefaction of ^99mTc-MIBI in the ischemia region where TTC was less uptake.The minipig with circumflex branch ligation was the better model for the myocardial infarction and circumflcx branch constriction for ischemia. ^99mTc-MIBI myocardial perfusion can define the infarction or ischemia segment in the living body while the TTC can also define in the isolated heart.

Ultrastructure of Murine Bone Marrow-derived Dendritic Cells

WU Shu-yan1, GU Tao2, ZHOU Huan2, WANG Xiang-ying1,LI Su-an1, MAO Di-hua1, ZHANG Xue-guang2

2004, 24(1):  34-36. 

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Dentritic cells（DCs）and their progenitors from murine bone marrow were collected and induced in vitro by both granulocyte-macrophage colony-stimulating （GM-CSF） and interleukin-4 （11,4） for 5?6 days. Immature DCs were loaded with apoptotic tumor cells （AP-DC），then AP-DC vaccines were induced further maturation stimulated with MCD40L-CHO cells and TNF-arespectively for 48 h. These DCs’ morphology was observed under transmission electron micro-scropy. We found that immature DCs showed a few short and blunt cytoplasmic processes,there were specific morphology granules which liked earphone in some cells；the immature DCs had a powerful ability to capture antigens from apoptotic tumor cells in 5-6 th culturing day，the DCs engulfing the apoptotic bodies were observed； sub-cellular structures between CD40 ligation and TNF-astimulated DCs were different, the former had typical morphology of DCs which exhibited many dendritic protrusions and the nucleus was irregular in shape，the cytoplasm contained numerous mitochondria, ribosomes，well-developed Golgi complexes, but scantly lysosomes. In addition,DCs might be in contact with each other. The results suggested that DCs were heterogeneous；DCs can acquire antigen by phagocytosing apoptotic bodies ；CD40 ligation and the associated signal transduction played an essential role in myeloid DCs differentiation and maturation.

Establishment of Orthotopic Heart Transplantation Model in Mini-pig

WU Li-hui，XU Zhi-fei，ZHAO Xue-wei，ZHANG Yu, FEI Yue-hua, JIN Yan

2004, 24(1):  37-38. 

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To establish heart transplantation model in mini-pigs under general anesthesia and ex-tracorporeal circulation, we used eight mini-pigs to harvest donor hearts for transplantation, with general anesthesia and extracorporeal circulation, excise recipient heart, and implant donor heart in original place. The hemodynamics was monitored during the operation. After transplantation all of the donor hearts beated again.The transplantation hearts withdrawn from cardiopulmonary circulation after 60min assistant extracorporeal circulation. The hemodynamics was stable. Establishment of heart transplant model in mini-pigs was successful.

Identification of Citrobacter freundii Caused Diarrhea in Rhesus Monkey

ZHOU Qin, GUO Guang-yuan

2004, 24(1):  39-40. 

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A strain of bacteria isolated from the feces sample of a bloody diarrhea rhesus monkey was identified to be Citrobater freundii by the Biomerieux methods of ID 32 E，ATB automatic analyzer，id%= 99.9.This strain is sensitive to norfloxacin，ciprofloxacin,cefotaxime,ceftriaxone,ceftazidime, chloramphenicol, and resistive to gentamicin, amikacin，tobramycin, flerox-acin，ampicillin，cephalexin, cefazolin，cephradine，trimethoprim/sulfamethoxazole, tetrecy-cline, and furazolidone by K-B method. So we should pay great attention to Citrobater freuyndii in isolating culture of rhesus feces.

Biological Characteristics and Application of BNX Mouse

FAN Shi-ming1，YAO Ming1，XING Hua2，LI Hou-da2

2004, 24(1):  50-54. 

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The origin and breeding of the immunodeficient animal——T、B、NK cell deficient mouse (BNX mouse) was reviewed and the biological characteristics and applications in haematol-ogy、oncology、immunology and other fields were given in details.

Establishment and Application of Rat Endometriosis Model

WANG Rui-xia, YU Chao-qin

2004, 24(1):  55-58. 

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At present,the pathogenic mechanisms of endometriosis are incompletely understood. Because the research in women is limited，the rat endometriosis model may be used to study events involved in the pathogenesis and pathophysiologies of endometriosis or novel therapeutic approaches for this disorder that are not accessible in human. This review is to discuss the application and value of the rat endometriosis model.