Table of Content

29 February 2008, Volume 28 Issue 2

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Establishment of a Highly Metastatic Model of Melanomain Mice in vivo Selection and Cell Line

WU Hai-yan1,2,YAO Ming2,YAN Ming-xia2,LIU Lei2,KONG Han-wei2,XUE Zheng-feng1

2008, 28(2):  67-73. 

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Objective To establish a highly metastatic model and cell line of melanoma B16 in mice by using organ selection of metastasis. Methods Histologically melanoma B16 intact tissues were subcutaneously implanted into the mice. In order to gain visible metastatic focus, BNX mice with immunodeficiency was selected according to the method of subcutaneous implantation → lung metastasis →subcutaneous inoculation → lung metastasis. In the first three generations neoplasma were removed by tumor resection when it reached 1.5 cm in diameter. In the following three generations the same steps were duplicated without resection. Then homologous cell line was established. Tumorgenicity, invasion, metastasis and morphological characteristics of the implanted tumors were studied by light microscopy, histology, cell growth curve, flow cytometry, Chromosome analysis and basement membrane invasion assay. Results Highly metastatic models of melanoma B16 were obtained after organ screening. After 3 generations in vivo, the carcinoma metastasis rate accounted to 80 percent, metastasis extent reached +++ after 45 days. While only a lower metastasis rate was found when the tumor was subcutaneously implanted absolutely without resection at the first time. But a 91.7 percent lung metastatic rate and high metastasis extent were observed again after being passaged in vivo for 3 generations after 35 days per generation. It indicated that B16-sci has similar characteristic with B16 by the assay about histology, cell growth curve, flow cytometry，Chromosome analysis and basement membrane invasion. Conclusions we had established a highly metastasis melanoma B16 model and homologous cell line， which was high, steady, and intuitionistic metastasis and have same biological characteristics of mice melanoma B16. The result provided a useful tool for the study of metastatic mechanism and treatment of carcinoma.

Establishment of Welfare Impaired Mice Model and Effect of Nutritional Intervention

YANG Fei,HU Ying,XU Lan-wen

2008, 28(2):  74-79. 

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Objective To establish an the welfare impaired mice model and investigate the effect of nutritional intervention. Methods Mice undergoing poor welfare due to repeated restraint ( simulate the procedures of laboratory routine) were used to measure the biological value when they were adapting to this procedure by testing welfare related indices (neuroendocrine parameters, immune parameters, biochemical parameters，body weight and food consumption ),and the effect of nutrient agar stick was assessed by using this model. Result Restraint mice showed higher basal serum CORT, ACTH, EPI, NE and lower basal serum [3-EP, IL-2 as well as lower body weight gain and FER compared with control group. Meanwhile，WBC, spleen index and splenocyte proliferation of restraint ones decreased significantly. Compared with model group, nutrient stick group showed lower levels of CORT, ACTH, EPI, NE, higher levels of [3-EP，IL-2, WBC, spleen index and splenocytes proliferation. All the difference was significant. Conclusion The study established successfully an animal model that display the neuroendocrine disorder，immune suppression and growth suppression due to impaired welfare, and suggested that nutritional intervention do benefit to improve the neuroendocrine disorder and immune suppression.

Modification of Suture Model of Focal Cerebral Ischemia in Rat

XIE Ting,ZHAO Yan,QU Hui-hua,Gao Wei-ping,LI Yi-fei,Wang Qing-guo

2008, 28(2):  80-84. 

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Objective To improve the rat model of suture occlusion of local cerebral ischemia. Method The right common carotid artery (CCA) and external carotid artery (ECA) were isolated afler anesthesia, We turned the rat head 45° from the middle line to the left, make ECA parallel to ICA and then inserted a thread with an enlarged tip 0.3 mm in diameter and 3 mm in length at the point 18 mm to the bifurcation of ECA and ICA. After 2 h the rats were assessed for neurological evaluation. 20h after MCAO，the rats were sacrificed for TTC stain, the volume was calculated. Result Neurological scoring of each model was 2 point. On TTC staining manifestations of cerebral infarction were found in all rats，and the volume of infarction was about(19.066±1.556)%，the brain volume ratio was about 1.031 ±0.024. The success rate was 93.75%, the mortality is 6.25% and the complication rate is 6.25%. Conclusion The model has shown to be easy to be performed and with high success rate so it is a useful animal model.

Antitumor Effect of Anthocyanidin on Lung Cancer NCI-H460 Cells in vitro and in vivo

GAO Ai-xia1,LUO Ju-dong2,WU Qing-ting3,LI Hou-da3,XUE Zhi-mou1

2008, 28(2):  85-89. 

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Objective To investigate the antitumor effect of anthocyanidin on lung cancer NCI-H460 cells in vitro and in vivo. Methods Cells proliferation and viability were analyzed by cells count and trypan blue exclusion assay，the inhibitory ratio of NCI-H460 cells was measured by MTT assay, the IC50 value was calculated, cells apoptosis were detected by DNA agarose gel electrophoresis, lung cancer cells NCI-H460 were subcutaneously injected into nude nice to establish cellular transplantation tumor nude mice model，and anthocyanidin were administered to the tumor-bearing nude mice,the kinetics of tumor formation and tumor growth were measured. Results After treatment of anthocyanidin, NCI-H460 cells viability decreased obviously, NCI-H460 cells proliferation were remarkably inhibited by different concentrations of anthocyanidin and the inhibition was both in time-and dose-dependent manner, the IC50 values were 90.8 jug/ml. The typical DNA ladder on agarose gel electrophoresis for analysis of cellular apoptosis were significantly appeared. Anthocyanidin can inhibit the tumor growth of nude mice. Conclusion Anthocyanidin can inhibit NCI-H460 cells proliferation, induce cell apoptosis and inhibit the tumor growth of nude mice.

Studies on Polymorphism of SLA-DQA Gene by PCR-RFLP in Tibet Mini-pigs

ZHANG Jian-ming1,CAO Gui-rong2,WANG Yu-jue1,LI Jin-ze1,YUE Min1,GU Wei-wang1

2008, 28(2):  90-94. 

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Objectives Analyzed genotype and allelic polymorphism of SLA-DQA gene extron 2 in Tibet mini-pigs to detect whether the genotype frequency of restriction enzyme sites was consistent with Hardy-Weinbeig equilibrium. Methods PCR-RFLP was used to analyze the partial sequences of intron 1 and intron 2 and the complete sequence of extron 2 of SLA-DQA in Tibet mini-pigs. Results The restriction enzyme map of EcoR I -RFLP site showed that the genotype frequency of BB(0.4500) was higher than that of AB(0.3167) and AA(0.2333), B was the predominant allele. As for the Alu I site, the genotype frequency of MN(0.5000) was higher than that of MM(0.3000) and NN(0.2000), and M was the predominant allele. According to the values of X2test (chi-square test), the genotypes at EcoR I site didn't reach the significance level(P＞0.05〕，while the Alu I site has significant differences (P＜0.05). Combined with double enzyme digestion result, there were 7 PCR-RFLP patterns of SLA-DQA gene in Tibet mini-pigs. The genotype frequency of BBMM(0.3000) was higher than other ones, while the genotype AANN or BBNN was not found. Conclusion The SLA-DQA gene of Tibet mini-pigs was not fit Hardy-Weinbeig equilibrium at Alu I site, and the genetic selection pressure of this site was higher than that of EcoR I site.

Pathological Observation on Chinese Rhesus Monkeys with Recombined Simian/Human Immunodeficiency Virus

HUANG Di1,HE Li-qun2,YANG Xue-jun2,XIAO Jing3

2008, 28(2):  95-98. 

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Objective To study pathological changes on Chinese rhesus monkeys with recombined Simian/Human Immunodeficiency virus. Methods Chinese rhesus monkeys were inoculated with a serial of four in vivo passages (P1-P4) of SHIV-B’ whu. Each serial were sacrificed on fourteenth day. Pathological changes of lymph nodes were studied respectively. Results The infected monkeys demonstrated a transient increase of body temperature during the acute stage of infection with all the passages of SHIV. SHIV-B’whu infection was also demonstrated by the pathological findings: focal necrosis in liver, interstitial pneumonia, partially enlarged lymph antrum, diminished lymph follicle, and erythrophagia in lymph nodes. Macrophage positive rates in monkey tissues increased at a uncertain gradient from PI to P4(from about 3.5% to 22%). Conclusion This study provide direct evidence that SHIV-B’whu has the ability to infect Chinese rhesus monkeys, which can present the similar symptom as human being.

Pathological Observation on Chinese Rhesus Monkeys with Recombined Simian/Human Immunodeficiency Virus

TANG Zhi-jiao,DAI Ming,BAO Rong,WANG Yong,ZHANG Jing,XIAO Qiao-yang,RAO Yan,LI Ren-xiang,SUN Li-hua

2008, 28(2):  99-101. 

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Objective To study pathological changes on Chinese rhesus monkeys with recombined Simian/Human Immunodeficiency virus. Methods Chinese rhesus monkeys were inoculated with a serial of four in vivo passages (P1-P4) of SHIV-B’ whu. Each serial were sacrificed on fourteenth day. Pathological changes of lymph nodes were studied respectively. Results The infected monkeys demonstrated a transient increase of body temperature during the acute stage of infection with all the passages of SHIV. SHIV-B’whu infection was also demonstrated by the pathological findings: focal necrosis in liver, interstitial pneumonia, partially enlarged lymph antrum, diminished lymph follicle, and erythrophagia in lymph nodes. Macrophage positive rates in monkey tissues increased at a uncertain gradient from PI to P4(from about 3.5% to 22%). Conclusion This study provide direct evidence that SHIV-B’whu has the ability to infect Chinese rhesus monkeys, which can present the similar symptom as human being.

Study on Radiotherapeutic Ovary Recovery after Bone Marrow Transplantation in Mouse

WANG Yu-jue1,CAO Gui-rong2,ZHANG Jian-ming1,Nashunbayaer1,GU Wei-wang1

2008, 28(2):  102-105. 

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Objectives To evaluate the effect of radiotherapic ovary recovery function by bone marrow transplantation and discuss the possible mechanisms. Methods One humdred female Kunming mice,at the age of 6?10 weeks were used，20 of them were selected as donor for bone marrow transplantation, 40 of them were selected as control group after once radiotherapy, 40 of them were selected as experimental group，processed transplantation 48 h after radiotherapy. On day 1st, 5th, 15th, 30th, the E₂, FSH level of these mouse were detected and the morphological changes of impaired ovary observed were. Results On day 15th and 30th，compared with control group, the experimental group presented more primordial follicle, primary oocytes，mature oocytes than control group, and showed less accrementition and hyalinization in ovarian vessel. The E₂ and FSH of control group was maintain at 18 ng/L and 3 IU/L，while the E₂ in experimental group was up to 24.69 ng/L, FSH level dropped to 2.14 mlU/ml. Conclusions The experimental group resumed generating new oocytes and follicles，the bone marrow transplantation may have recovery function to radiotherapic impaired ovary.

Changes of Serum Levels of Estrogen and Progestogen during Menstrual Cycle in Normal Female Cynomolgus Monkeys

MA Nan-hua1,LI Zong-qiang2,QIN Jian1,ZHENG Li-ping1,ZHU Xu1,HUANG Sheng-xian1,XIE Li-ping3

2008, 28(2):  106-109. 

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Objective To observe the changes of estrogen and progestogen during the menstrual cycle in normal female cynomolgus monkeys. Methods Twenty sexually mature female cynomolgus monkeys aged 4-6 years and weighed 3.4-8.4 kg were enrolled in this study. Their blood specimens were collected regularly every day for 31 ?51 days to determine the serum levels of estradiol (E2) and progesterone (P) by microparticle enzyme immunoassay. Besides，the changes of E2 and P during the menstrual period，follicular phase, ovulatory period and luteal phase were observed in 6 of these monkeys with distinct menstrual cycle. Results The serum levels of E2 and P changed along with the menstrual cycle. The peak values of E2 occurred during the ovulatory period as well as luteal phase with the highest value during the ovulatory period; the peak values of P occurred during the luteal phase. Conclusion The changes of estrogen and progestogen during the menstrual cycle in normal female cynomolgus monkeys are similar to those in women of childbearing age. Therefore, normal female cynomolgus monkeys may be taken as models to study the changes of estrogen and progestogen levels in various stages of menstrual cycle in women.

Modification and Comparison of Two Methods for Creating Rats Models of Acute Necrotizing Pancreatitis

LIU Na-xin1,GUAN Min-qiang2,CHEN Tong-ke2,ZHAO Hui-ling2,CHEN Xi-wen2

2008, 28(2):  110-113. 

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Objective Compare two methods for creating the rat model of acute necrotizing pancreatitis and their application. Methods Fourty SD rats were randomly divided into biliopancreatic duct directly punctured group (A group，n =16)，duodenal wall punctured group (B group, n=16) and control group (C group，n = 8). One-half of the animals of A group and B group was sacrificed 24 h after operation (as A1 group and B1 group), and the following parameters were examined: the model creating time, the volume of ascetic fluid, serum amylase and the severity of pancreatitis. Four days after operation, survival analysis of the remaining rats in A group and B group (as A2 group and B2 group) was performed. Results The histological manifestation of acute necrotizing pancreatitis was observed 24 h afler operation in A1 group and B1 group; The serum amylase and the ascetic volume of A1 group and B1 were higher obviously than C group (P<0.01), and these parameters were no different between A1 group and B1 group; The model creating time between A1 group and B1 group were different (p<0.05)，and the survival rate was significantly higher in B2 group than that in A2 group(p<0.05). Conclusion The method of puncturing duodenal wall is superior to that of directly puncturing biliopancreatic duct for creating acute necrotizing pancreatitis; while the later can be used in some short term studies because of its simple manipulation.

Establishment of Polymerase Chain Reaction for Detection of Pseudorabies Virus in Pigs

LI Chun-hua1,HU Jian-hua2,ZHANG Wan-hua1,SUN Feng-ping1,GAO Jun1,WANG Ying1,JIANG Feng-ying1

2008, 28(2):  114-117. 

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Objective To establish the method of polymerase chain reaction (PCR) for detection of the pseudo rabies vims (PrV) in pigs. Methods A pair of primers were designed and synthesized according to the sequence of gB gene of PrV. PCR method for PrV was established by optimizing the reaction parameters，and utilized for detection of PrV m clinical samples. Result The 263 bp product was amplified specifically by using the DNA of PrV SH strain as template. The amplified product was cloned into pMDl 8-T vector and sequenced. The identity of nucleotide sequence of this fragment and gB gene of genbank was 99.2%. The identical results were obtained by PCR and virus isolation of clinical samples. Conclusion The PCR method established is sensitive and specific for the rapid detection of PrV in pigs.

Effect of in vitro Fertilization on Early-embryo Development and Serum Biochemistry in Sexual Maturity Mouse

FENG Yu-fang1,TAO Ling-yun2,3,SHAO Wei-juan4,GAO Cheng2,3

2008, 28(2):  118-120. 

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Objective To evaluate the effect of in vitro fertilization (IVF) on early embryo development and serum biochemistry in sexual maturity mouse. Methods The 2-cell embryos from IVF were transferred into pseudo-pregnant recipient mice. The fetus were weighed afler the embryo developed to 14 days (El4).Meanwhile，several pregnant mice were maintained until the pups were bom and weighed. The newborns were raised until sexual maturity, and the serum biochemistry of the mice was analyzed. Result The weight of E14 fetus from IVF group was 16.5% higher than that of the control group (P＜0.001)，the birth weight of IVF group was 13.2% higher than that of control group (P＜0.001)，but the differences of serum biochemistry in sexual maturity mouse between IVF group and control group were not significant. Conclusion The in vitro fertilization can affect the mouse early-embryo development, but it doesn’t affect the serum biochemistry in sexual maturity mouse.

Observation on Hippocampal Cellular Oxidative Damage Induced by Different Concentration of H₂O₂ in Rats

DIAO Bo,TANG Ying,WANG Xiao-kun,LI De-zhong

2008, 28(2):  121-124. 

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Objective To explore the best concentration of HzOz to induce hippocampal cellular oxidative damage in rats. Method The hippocampal cell from the rat was co-cultured with H₂O₂ in diversity concentration for 24 hours. The changes of the cell morphology was observed, the viability of hippocampal neures was detected with MTT method; the expression of Bax and Bcl-2 were detected by immunohistochemical method. Result Compared with normal group, when the concentration of H₂O₂ is higher than lmmol/L,the morphology damage of hippocampal cell is obviously aggravated, the cytoactive is low,the Bax is high expression. When the concentration of H₂O₂ is lower than lmmol/L, the morphology damage of hippocampal cellular is not obviously aggravated, the cytoactive has no obvious change, the Bcl-2 is high expression. Conclusion The best concentration of H₂O₂ to establish cell oxidative damage model is lmmol/L.

The Anatomic Feature of Rabbit Tibia Medullary Cavity and Designing of Intramedullary Nailling Faxition Model

XUE Feng1,HOU Chun-lin2,CHEN Qing-quan2

2008, 28(2):  125-128. 

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Objective The purpose of this study was to investigate the anatomic parameter of the rabbit tibia, and to design intramedullary nailing model on the rabbit tibia fracture faxition according to the medullary cavity anatomic feature. Method Eighteen adult New Zealand white rabbits (2.8?3.0 kg) were used in the study. Both sides of the tibia were harvested and transverse computed tomographic scanning was conducted in the interval of every 5 mm and the internal diameters were messured. The intramedullary nail for tibia fracture faxition was designed according to the rabbit tibia medullary cavity anatomic feature. The osteotomy was conducted at the board of upper and middle part of tibia. The models of tibia fracture intramedullary faxition were done. Result The shape in medullary cavity transverse section of upper part of the tibia was triangular. That of the lower part was oval. While the middle part was the transform of the two shapes. The intramedullary nail was designed according to the anatomic characteristics with it，s transverse section being triangular. The fixation was satisfactory. Conclution The intramedullary nail designed according the anatomic feature of the rabbit tibia medullary cavity and the intramedullary nailing model on the rabbit tibia fracture faxition was rational.