Table of Content

30 April 2010, Volume 30 Issue 4

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The Targeting Study of Rabbit HGPRT Gene

WANG Wei1，ZHANG Chuan-shan2，GUO Yi3，GU Rui-huan3，DING Lei4，YAO Gang1，CHEN Xue-jin3

2010, 30(4):  233-240. 

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Objective To construct a rabbit HPRT gene targeting vector. Methods The rabbit full length HGPRT gene BAC clone LBNL1-304M19 is used as the template. A 12.5kb rabbit HGPRT gene fragment, which does not include promoter，is cloned into pKS- plasmid to form pKS-HGPRT recombi-nant plasmid via Gap-Repair by Red recombination system. Then we construct a GFP and neo selective targeting vector pKS-HGPRT-GFP-neo on the basis of pKS-HGPRT and pEGFP-Cl-SD1211 plasmids.Results Linearized targeting construct DNA was introduced into the rabbit fibroblasts by Lipofectamine^TM 2000. The positive-negative selection was performed and survival clones were screened by PCR and sequencing, and eight colonies with homologous recombination were obtained. The targeted colonies would further confirmed by Southern blotting. Constructions The knockout HPRT targeting vector was rapidly constructed and the results set a good basis for the establishment of HGPRT-knockout rabbit by gene target and nuclear transfer methods.

Preliminary Study on Conditions of Transfection of Rabbit Adult Fibroblasts by Electroporation

GU Rui-huan1，LI Shan-gang1，SONG Wei-jie2，WANG Wei3，DING Lei2，ZHANG Yan1，XING Feng-ying1，LI Yao1，CHEN Xue-jin1

2010, 30(4):  241-245. 

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Objective To investigate and explore conditions of tansfection on the rabbit adult fibroblasts by means of electroporation, the plasmid vector of pEGFP-cl was successfully transfected into the rabbit adult fibroblasts by electroporation. Methods The rabbit fibroblasts at 7-10 passages were transferred with pEGFP-cl by means of electroporation with different electric voltages, capacitors，the doses of DNA, different incubation time before electroporation, incubation temperature after electroporation. The cell survival rate was counted under visible light microscope, and the transfection efficiency and production of protein were identified through FACS analysis. Results transfection efficiency and cell survival rate were of the higher value, when The rabbit fibroblast cell with concentra-tion of 2 × 10⁶cells/ml (electroporation medium: PBS) and mixed with 30 jig vector DNA，the electric voltage of electropration apparatus was set at 250 V, and the capacitors was 700 jiiF. Moreover，the electransfected efficiency and cell survival rate was increased with 10 min of ice incubation before electroporation and 10 min of 37℃ incubation after electroporation. Conclusion The electrotransfection is very critical for the tansfection on the rabbit adult fibroblasts，and incubation temperature could improve transfection efficiency.

Construction of Eukaryotic Expression Plasmid for 5-Lipoxygenase Gene and its Expression in RAW264.7 Cell

WU Hong-lian, ZHANG Mei-ying，WANG Wei

2010, 30(4):  246-250. 

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Objective To construct 5-Lipoxygenase gene eukaryotic expression vector and study its expression in R AW264.7cell. Methods The gene of 5-LO was amplified from human peripheral blood by reverse trameriptage-pelymerase chain reaction(RT-PCR)，and then inserted it into cloning vector pUCm-T，and eukaryotic expressing vector pEGFP-C2 respectively. Recombinant plasmids were trans-formed and screened，and identified by PCR, restriction enzyme digesdon and DNA sequencing. The recombinant epressing plasmid 5-LO was transfected into RAW264.7 cells. Then detected the expres-sion of the interesting gene 5-LO. Result The recombinant plasmid pUCm-5-LO and pEGFP-5-LO were successfully constructed, and the correct sequence of 5-LO identified by PCR, restriction enzyme, digestion and DNA sequencing. The recombinant expressing plasmid pEGFP-5-LO was successfully transfected into RAW264.7 cells and it could be effectively expressed which was also testified by RT-PCR and Western blot. Conclusion The recombinant eukaryotic expressing plasmid ofpEGFP-5-LO is successfully constructed and effectively expressed in RAW264.7 cells，and it may be useful for further research of 5-LO transgenic mice.

The Preliminary Study on Correlation of Experimental Acid Aspiration and Pulmonary Fibrosis in Rats

CHEN Shi1，ZHANG De-ping2

2010, 30(4):  251-256. 

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Objective To study the pathology and possible mechanism of pulmonary fibrosis in rats induced by experimental hydrochloric acid (HCl) inhalation and compared with traditional bleomycin-induced fibrosis. Method One-hundred and twenty male SD rats were randomly divided into nomal control, bleomycin，high，middle and low-dose HC1 group.The bleomycin group was intratracheally injected bleomycin, the HC1 groups were intratracheally injected HC1 once per week, and the control group was given saline in the same way.Six rats of each group were randomly sacrificed on day 7，14，28 and 42 respectively. The histological changes of the lung tissue were evaluated by HE and Masson's trichrome staining. The mRNA expression of transforming growth factor-β＜sub＞1＜/sub＞(TGF-β＜sub＞1＜/sub＞) were assayed by RT-PCR. The CTGF expression were analyzed by immunohistochemistry. Results Hydrochloride groups has significantly higher alveolitis levels (P＜0.01) and can reach or exceed the level of bleomycin group after 28d. Fibrosis in hydrochloric acid groups were increased gradually over time，but not consistently exceeded bleomycin group (P＞0.05). Similar results appear in the TGF-β₁ mRNA and CTGFexpression in immunohistochemical identification. Conclusion The results revealed the relationship between the gastroesophageal reflux disease (GERD) and idiopathic pulmonary fibrosis (IPF).

Observation on Characteristic Changes of Acute Lung Injury Induced by PFID Inhalation in Rats

WU Shan-shan1，LIU Ji-fu1，LIANG Hai-long1，ZHANG Xian-cheng2，HUANG Chun-qian2，ZHAO Jian2

2010, 30(4):  257-260. 

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Objective To establish the model of acute lung injury(ALI) of perfluoroisobutylene (PFIB) inhalation and to study the change of the cell factor in blood and lungs tissue，pathological changes of the lung tissue. Methods The model of acute lung injury was induced by (PFIB) inhalation.The protein concentration in bronchoalveolar lavage fluid (DALF), the liquid volume of lung, and the cell factor of interleukin-1、8、10、18 and TNF-a were assessed. The pathology of lung injury were examined. Rusult (l)In the model rats, the liquid volume of lung, and BALF protein concentration are higher than those of negative group. (2) In the acute lung injury rats of PFIB, the characteristic changes of the interleukin -1、8、10、18 and TNF-a in blood and lungs tissue were deteted; (3) The pathology shows that the changes of pulmonary edema and the inflammayory reaction of ALI. Conclusion The research and exploration on the changes and regularity of ALI induced by PFEB inhalation can provide more information about ALI protection and treatment.

Preliminary Study on Correlation between Plasma Titers of Ox-LDL Auto-antibody and Extent of Atherosclerosis in Mice Auto-antibody and Extent of Atherosclerosis in Mice

CHEN Jia-wei，ZHOU Shi-bei，TAN Zhi-ming

2010, 30(4):  261-264. 

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Objective To identify a potential plasma marker for the prediction of atherosclerosis in mice. Methods C57BL/6J mice were fed on regular diet. Apo-E deficient mice (on C57BL/6J background) were divided into four groups，group A: high cholesterol diet (1% cholesterol) alone, group B: high-cholesterol diet with rosuvastatin (lmg/kg/d)，group C: high-cholesterol diet with candesartan (1 mg/kg/d)， and group D: high-cholesterol diet with rosuvastatin and candesartan. All the mice groups were fed for 12 weeks. Plasma titers of ox-LDL auto-antibodies in relation to the extent of atherosclerosis in these mice were measured. Results C57BL/6J mice had no atherosclerosis, whereas mice in group A had extensive atherosclerosis. The extent of atherosclerosis was decreased in group B and C mice, despite high-cholesterol diet. Moreover, the extent of atherosclerosis was further decreased in group D mice. The plasma titers of four ox-LDL auto-antibodies were measured. It was found that the mean values of all antibodies, includ-ing Cu-LDL-IgG, Cu-LDL-IgM，MDA-LDL-IgG and MDA-LDL-IgM, were higher in group A mice than in C57BL/6J mice (P＜0.01). Compared with group A mice，group B，C and D mice had lower plasma titers of Cu-LDL-IgG, which was consistent with the decrease in extent of atherosclerosis. Compared with that of C57BL/6J mice, plasma level of ox-LDL reduced in group A mice (P＜0.01). Although plasma level of ox-LDL also reduced in group B and C mice，it was restored back to normal level in group D mice. Conclusion The plasma titer of Cu-LDL-IgG is consistent with the progression and regression of atherosclerosis and it may serve as a marker for the prediction of atherosclerosis.

Pattern Characteristics and Efficacy Evaluation of Treatment Based on Pattern Identification of GK Rats

LIU Xiao-mei，FANG Zhao-qin，LU Wen-li，PAN Zhi-qiang，GUAN Dong-yuan，LIANG Chao

2010, 30(4):  265-268. 

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Objective To characterize GK rats as well as the efficacy of treatment based on pattern identification. Methods GK rats were randomly divided into model group, western medicine group and integrated Chinese and western medicine group. Wistar rats were set up as normal group. All rats were provided water or corresponding medicine. The change of random blood sugar and manifestations of four diagnosis were assessed. Results The efficacy of individual syndrome differentiation and treat-ment on lowering stomach energy and random blood sugar is better than simple western medicine.Moreover, syndrome differentiation and treatment can slightly rcduce yin deficicncy and periodically affect qi deficiency. Conclusion GK rats have particular syndromes and accompanying symptoms which progress regularly. Individual syndrome differentiation and treatment have certain dominant position.The efficacy of which has wide space to explore.

Establishment of a Cyclomastopathy Model of Stagnation of Hepatic Qi in SD Rats

WU Shu-guang1，WU Pei-xin2，WANG Ming-zhen1，CHEN Zhen-zhen1，ZHANG Shu-sen1，LI Shu-chai1，QIAN Ning1

2010, 30(4):  269-272. 

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Objective To establish a rat cyclomastopathy model of combination of disease and syndrome. Methods Hormone combined with restraint method is used in this study. The Estradiol Benztrone was given to rats by intramuscular injection(l .0 mg/kg) for 10 times，one time every three days. Then, the progesterone injection(5.0 mg/kg) was used for 5 days. And the rats were restrainted during the night for 15 limes, performed every two days. The day after the final injection, mammilla diameter was measured, open-field behaviors were observed. Blood and mammary gland were taken for further test in all the rats. Results In model rats group, mammilla was intumescent. Both vertical and horizontal movements were reduced. Iligh-leveled prolactin and estrodiol accompanied with low proges-terone were found in blood serum. Increase in number and size of lobules of mammary did not recover to the normal level in three weeks. Conclusions Hormone combined restraint method is easy to operate to establish a duplicate rat cyclomastopathy model successfully, and it is in accordance with the clinical pathogenesis.

Exploration on Application of Aerosol Delivery of Mycobacterium smegmatis for Creating a Animal Model of Pulmonary Infection

LIU Fang1，YANG Hua1，Zhou Wen-jiang2，Fan Xiao-yong1

2010, 30(4):  273-276. 

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Objective In order to establish the aerosol bacteria delivery method applied to the animal model of Pulmonary infection, a non-toxic fast-growing Mycobacterium smegmatis was used as a model bacteria and the effects, influence factors and safety about this technology was investigated.Method The C57BL/6J mice were infected by mycobacterium smegmatis at a series of dose levels (10⁶～108 CFU) and duration (30-90 min), an auto aerosol delivery device were used for aerosol bacteria preparation and infection procedure. After infectionL samples from lung tissue and skin of infected mice as well as samples from surface of the device were obtained and then cultured for bacteria amount counting by CFU method. Result The standard curve of bacteria amount-dose level and bacteria amount-infection duration were respectively set up. Existence of residual bacteria in device surface and skin of the animals was indicated by bacteria amount counting results (less than 10 CFU). Conclusion The bacteria amount in mice' lung tissue is directly and positively correlated to the dose level of aerosol bacteria and infection duration and existence of residual bacteria points out potential bio-hazard risk.

Effect of Lighting Asymmetry on Blood Physiological and Biochemical Indexes in SD Rat

WANG De-jun，ZHOU Wei-min，WANG Hui，SHOU Qi-yang，ZHU Ke-yan，CHEN Min-li

2010, 30(4):  277-282. 

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Objective To observe the effect of lighting asymmetry on the blood physiological and biochemical indexes of normal SD rats. Mothods Eighty SD rats, weighting 60～80 g，were kept on the rack with different layer (A,B,C,D) after one-week's adaption . Light-intensity and light area were measucrd in each cage. Food consumption and body weight were calculated every 2 weeks. Blood physiological and biochemical indexes were detected after 8 weeks. Results (l)The light-intensity among inner inside of four layer's cages was no significant difference (P＞0.05)，however, there is a significant difference light-intensity and cage-area in outside and median site of cages in each layer (P＜0.01, P＜0.05).(2)The food consumption and weights of A and D layers rats have no obvious differences (P＞0.05).(3)The blood physiology shows WBC,MCHC, HDW, MPV, NEUT, EOS, BASO and Retic of female rats from A to D layers varied at different degrees (P＜0.05,P＜0.01). (4) The Blood Biochemistry shows AST, TBIL, UA, TG，CK, and CK-MB of female rats from A to D have obviously decreased, ALP and GLU have significantly increased (P ＜0.05，P＜0.01); while TBIL and CK-MB of male rats were markedly decreased, GLU and BUN were notably enhanced (P＜0.05, P＜0.01). (5) Female rats compared with the males on the same layer, some physiological and biochemical indicators of blood there were significant differences(P ＜0.05, P ＜0.01). Conclusion The light asymmetry was in different layer of the cages can cause the change of blood biochemical indexes to a certain extent, and the change was more obvious in female rats.

Experimentation of the Second Absorbable Complex Enhancement Bolt for Mechanical Properties,Biodegradation and Biocompatibility

ZHONG Zhen-dong1，ZHANG Xiao-jin2，ZHONG Hao1，WANG Kai-yu3，CHEN Jin2，LV BO4，YANG Rong-fang4，YAN Na1

2010, 30(4):  283-287. 

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Objective To evaluated the mechanical properties, biodegradation and biocompatibility of the Second Absorbable Complex Enhancement Bolt by animal experiment. Methods Based on the Beagle dog model for fracture of femur in 12 weeks，the bolt's bending strength, and its rate of decay,and biological absorption rate (BAR) were observed, the fracture fixation，and the growth of bone trabe-cula and bony callus were determined by Using X ray, microscope and electron microscope，and the Second Absorbable Complex Enhancement Bolt was compared with the First one at bone formation in experimental animal. Results The bending strength of the Second Absorbable Complex Enhancement Bolt was stronger than the First one in the second, fourth, eighth, twelfth week，and its rate of decay was lower than the First one. Two Bolt's bending strength decreased fast in the forth eight weeks. After that time, the decay was stabilization, and its profited the union of fracture and remodeling. At the same time，Two Bolt's biological absorption rate had no difference in statistical significance. Compared with the first bolt，the second one was earlier to promote necrotic tissue, fibroplasias，thready and osseous callus formation, and the result of X ray was hinted that bone fracture was henosised earlier. Conclusion The Second Absorbable Complex Enhancement Bolt could satisfied the need of the mechanical strength for bone fracture ’s internal fixation materials, and profit the thready and osseous callus formation.

Preparation of Monoclonal Antibody Against Human 4-1BBL

CHAI Qing，GAO Wen-jie，XIE Wei，PAN Jian-zhong，JU Song-guang，ZHANG Xue-guang，XUE Zhi-mou

2010, 30(4):  289-292. 

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Objective To prepare monoclonal antibodies against human 4-1BBL molecule and analyze their biological characteristics. Methods The BALB/c mice were immunized with the cell line L929/4-1 BBL. The splenocytes from the immunized mice were fused with SP2/0, and then the hybridoma cells were screened with immunostaing and FACS. The isotype of the monoclonal antibody was identified by rapid isotyping analysis, and the 4-1 BBL expression profile on tumor cell lines was analyzed by indirect immunofluorescence with obtained mAb 39A8. Furthermore, the myeloid leukemia cell line U937 cells were stimulated with mAb 39A8 and their proliferation was measured with cell number counting. Results A hybridoma cell line named 39A8 was obtained, which had the property of secreting anti-human 4-1 BBL monoclonal antibody continuously and stably. The mAb 39A8 could recognize 4-1 BBL molecules on different cell lines and could enhance the proliferation of U937 cells in dose-dependent manner. Conclusion This work established an agonistic anti-human 4-1BBL mAb which may provide a useful tool for studying 4-1 BBL and its biological function.

Preparation of Neutralized Mouse Anti-human FL Monoclonal Antibody

GAO Wen-jie，CHAI Qing，ZHANG Xue-guang，JU Song-guang，XUE Zhi-mou

2010, 30(4):  293-296. 

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Objective To prepare monoclonal antibodies against human FL molecule and analysis of their biological characteristics. Methods The BALB/c mice were immunized with the transfected cell line L929/FL that expressed membrane FL. The splenocytes from the immunized mice were fused with myeloma cell line SP2/0，and then the hybridoma cells were screened with immunostaing and FCM. The isotype of the monoclonal antibody identified by rapid isotyping analysis, and the FL expression profile on tumor cell lines was analyzed by indirect immunofluorescence with mAb 3E5. To analysis the biological function of mAb 3E5, we added the 3E5 to the RPMI1640 medium containing FL protein to culture THP-1 cells. Results a hybridoma cell line named 3E5 was obtained, which had the property of secreting anti-human FL monoclonal antibody continuously and steadily. Fast-strip method analysis displayed that these mAbs belong to mouse IgGl，K. The mAb 3E5 could recognize FL molecules on different cell lines. FL protein could enhance the proliferation of THP-1, and it could be neutralized by 3E5 mAb. Conclusion Our study established a neutralized anti-human FL mAb which may prove a useful tool for studying the biological fUnction of FL.

Progress in Research of Animal Models with Melamine Urolithiasis

ZOU Yi-hai1，ZHANG Wei2，GUO Xue-jun1，XIE Ling-ling1，CHE Jia1

2010, 30(4):  308-308. 

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The melamine mainly applies as the industrial chemicals in the industrial production，if is increased to food, may cause the urolithiasis, serious may lethal therefore the use melamine development urolithiasis animal model，to carries on its mechanism and the treatment research has the vital practical significance, the article from the melamine physics and chemistry nature, metabolism, the toxicity, the symptom of poisoning，the toxicant mechanism as well as the recent years melamine induced the person and the animal urolithiasis experiment report and so on is various, summarizes the melamine urolithiasis animal model study to progress, and further proposed the Chinese medicine in the prevention urolithiasis aspect's function, will be beneficial in the melamine urolithiasis stone model more selective research.