Table of Content

25 December 2002, Volume 22 Issue 4

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Preparation of the Anti-PEA Monoclone Antibody and the Cloning of Its ScFv

CUI Shu-fang, TANG Qiu,HAO Guan-grong, CAO Ping

2002, 22(4):  199-203. 

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To get the single chain variable fragment antibody （ScFv） gene of active higher the monoclonal antibody cell 2F6 with higher neutralizing activity，the hybridoma cell lines 4D5、2F6 and 7G3, which could secrete anti-PEA monoclone antibody were prepared. The V_H、V_Lgene were amplified from the cell line 2F6 by RT-PCR. The V_u and V_L gene were connected through a flexible linker （Gly4Ser） 3, and the V_H-Linker-V_L （ScFv） fusion gene was cloned into a vector pGEM-T.The ScFv gene was sequenced, analyzed by computer and the results were showed that it was consist of 729bp ,encoding 243 amino acid residues. Both V_H and V_L were confirmed as functionally rearranged mouse immunoglobulin variable region genes and appeared to be new genes,According Kabat classed method , McAb 2F6 V_H gene segment and V_L gene segment belong to the mouse Ig heavy chain subgroup Ⅱ（A） and κ chain Ⅵ subgroup respectively.

A Convenient and Rapid Model in vitro to Screen Steroid 5 alpha-reductase Inhibitors

SUN Zu-Yue, WU Jian-Hui, TU Zeng-Hong, ZHU Yan, ZHONG 恩Hong

2002, 22(4):  204-208. 

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A convenient and rapid model in vitro to screen steroid 5α-reductase inhibitors which were effective in the treatment of benign prostatic hyperplasia （BPH） was developed. In the attendance of nicotinamide adenine dinucleotide phosphare （NADPH）, steroid 5α-reductase converts testosterone to dihydrotestosterone which is a major etio-logic factor of BPH.NADPH has characteristic absorbance at 340nmol/L，and the absorbance spectrum may be used to identify NADPH as a kind of the substrate in this enzymatic reaction. In this paper, NADPH, steroid 5α-reductase，series concentration of testosterone and finasteride, and 4 ml Tris-HCl buffer were continuously incubated together at 37°C and the NADPH OD value were continually measured.The descending rate of NADPH OD340 nm value by linear regression from the beginning to the tenth minute is close to the initial velocity of the enzymatic reaction. The precise activity of the steroid 5a-reductase was the slope after subtracted the one of the blank control. The inhibition constant（Ki）of steroid 5α-reductase inhibitors could be calculated according to the Lineweaver-Burk plots. Two drug screen models, the common isotope model and this novel model，were compared in this paper. The result showed that the latter one is more economical, quicker and more effective than the former one.

Relationship between the Inserted Thread Length and the MCA Origin in a Rat model of MCAO Made by Thread Occlusion Method

XU Jia, GE Lin-bao, XU Ming-shu , CHEN Han-ping

2002, 22(4):  209-212. 

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To determine the distance between the middle cerebral artery （MCA） and the origin of internal artery （ICA） in rats and observe the relationships of the inserted thread length to the MCA origin and to the successful model-making rate.：A thread occlusion method was used to make a MCAO model. Artery perfusion color casting was performed，and the distance between the origin of MCA and the origin of ICA was measured in the inserted thread length group. The cerebral infarct area was analysised and the cerebral infarct volume was calculated by computer image processing in the infarct volume group. The result showed that the distance between MCA and ICA ranged from 1.55cm to 1.65 cm. On the condition that the length of the inserted nylon thread was less than or equal to 1.60 cm, the successful model-making rate was 28.57%） if the length was more than 1.60cm , the successful rate was 83.33% （P＜0.05）. Conclusion：If we use a thread occlusion method to make a rat MCAO model, the length of inserted nylon thread must be no less than 1.65cm.

Comparative Study on the Vasodilating Effect of Diamox and Dipyridamole on Abdominal Aorta in Rabbit

MA Hong-Xing, XU Lan-Wen, CHEN Shao-Liang

2002, 22(4):  213-217. 

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To investigate whether Diamox （Acetazolamide） or dipyridamole can dilate the abdominal aortic ring in rabbit directly and its mechanism also, six rabbits with the body weight of 2.33±0.18 kg were anaesthetized and the aortic rings about 5mm long were quickly excised and emerged in oxygen saturated Kreb’s solution. The aortic ring was connected to a force transducer by one thread and fixed by another. The tension signal was transmitted to and analyzed by a microcomputer. After 10 minutes of stabiliza-tion ,the preload was set at 1.96×10^-2 N and force signal was recoreded. Then the aor-tic ring was contracted with norepinephrine. Fifteen minutes later, 15 mg of Diamox or dipyridamole was given. If vasodilatation was observed, 0.045g of sodium iodide was given 15 minutes after norepinephrine. Besides this, N-omega-L-arginine-methyl-evSter, glibenclamide，clotrimazole or indomethacin was given before norepinephrine,and Diamox was given 15 minutes after norepinephrine. The results showed that norepinephrine induced rise in force did not change during the 30 minutes observation period. Diamox significantly dilated the aortic ring but sodium iodide did not. Dipyridamole did not influence the tension of aortic ring. Clotrimazole significantly abolished the vasodilating effect of Diamox. Nw-nitro-L-arginine-methy 1-ester, glibenclamide or indomethacin did not influence the vasodilating effect of Diamox. Conclusions：Diamox can directly dilate the rabbit’s aortic ring. Dipyridamole does not have any direct vasodilating effect on the rabbit’s abdominal aorta.

The Effect of KangXianLing Decoction on Hemodynamics in Rats after Ischemia-reperfusion

HUANG Zhong-di, HE Li-qun, ZHANG Chang-ming

2002, 22(4):  218-222. 

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To approach the effect of KangXianLing decoction and its main-herb on the intervention of hemodynamics in rats after ischemic-reperfusion. The male SD rats were divided into 5 group （normal，sham-operated，experimental model, ZhiDaHuang , Dan-Shen, Monopril and KangXianLing group. The changes of plasma blood urea nitrogen （BUN）、serum creatinine （Scr） , endothelin-1 （ET）、thromboxance B₂ （TXB_2>）、 6-Keto-Prostaglandin-F_lα（6_Keto-PGF_lα）、nitric oxide synthase（NOS）, plasma and renal-tissue nitric oxide （NO） were detedted in each group after ischemic-reperfusion. The results showed that KangXianLing decoction and its main-herb significantly made BUN、Scr re-duced （P＜0.05），adjusted synthesing ratio of thromboxance A₂ （TXA₂）and prostacy-clin（PGI₂）,stabilized the content of plasma NO、NOS and renal-tissue NO. Conclusion：Kang XianLing decoction can remit the disorder of hemodynamics by I-R. So it can prevent renal function injury from renal ischemia.

Culture, Cryopreservation and Recovery of Adult Buffalo Lens Epithelial Cells

HU Jian-shi，WENG Jing-lin，ZHENG Zhi-liong

2002, 22(4):  223-226. 

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The modified method for cultivating an adult buffalo lens epithelial cells was described. The cell growth in vitro and the recovery of cells from frozen stale was investigated by cell counting , MTT and transmission electron microscopy. The results appeared that there is no significant difference in growth between cells cultured in vitro and recovered from frozen stale （P＞0.05），with characteristic and structure similar to the normal epithelial cell. The results suggested that planting culture is a kind of culture method for lens epithelial cells and freezing is an effective method to keep in cryopreservation such cells.

The Effect of Low Calcium Diet on Histomorphology of Tibia in Rats

MO Mei-ying,CHEN Jian-bo ,LI Qing-nan, LENG Yang,HUANG Liang-fan

2002, 22(4):  227-230. 

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To compare the effects of low calcium diet on bone in rats by bone histomor-phometry. Thirty-six 3-month-old Sprague-Dawley male and female rats （each half） were randomly divided into three groups, control group, very low calcium diet group （VLCD） and low calcium diet group （LCD）. After 90 days, all rats were killed. The proximal tibia （PTM） was harvested for histomorphometric analysis. The results revealed that the VLCD group male and female rats had lost 57% （P＜0.01） and 40% （P＜0.05） of cancellous bone compared with that of control group by 3 months respectively. Dynamic data in male and female rats showed that bone formation indices such as percent labeled perimeter（L. Pm%）,mineral apposition rate （MAR） and bone formation rate（BFR/BV） all increased without significance. Bone resorption indices such as osteoclast number per mm （Oc.N/Tb.Pm） increased 63% （P＜0.01）and 56% （P＜0.01）. The effect on LCD was lessen than in VLCD and there is significant bone loss only in male rats. The results indicated that bone loss was associated with increased bone turnover in low calcium dietary rats. The male rats were more sensitive to low calcium diet than female rats.

Hepatic Cirrhosis Model of Animal

ZHOU Zhong-xin1，WANG Jie2

2002, 22(4):  245-248. 

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Cirrhosis model （CM） of animal was the base for cirrhosis, portal hypertension and hepatocarcinoma research in human. Monofactor method was generally used to make CM of specific etiological factor. Multiple factors method was used to increase success rate, reduce time consuming and death rate. The problem at present were no CM of hepatitis , fewer reports on large animals , harmful inducers to researchers，low successful rate and high death rate.

Bacterial Infection and the Apoptosis of Mouse Macrophage

ZHOU Zheng-yu1, WU Shu-yan2, XUE Zhi-mou1

2002, 22(4):  249-251. 

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Bacteria can induce apoptosis of the mouse macrophage through its virulent substances such as endotoxin , exotoxin, invasin etc. This is a strategy used by bacterial pathogens to overcome host defense. The changes of morphology and biochemistry or biophysical characters of its membrane may be occurred in apoptosic macrophage. The apoptosic macrophage elicit an inflammaton by secretion of proinflammatory cytokines , which is an important factor causing infection limited. The study on the interaction between bacterial and macrophage apoptosis would be important in the development of the therapy and prevention of infectious diseases. We reviewed the advance in the mechanism and checking methods of mouse macrophage apoptosis induced by bacterial infection.