Table of Content

25 February 2013, Volume 33 Issue 1

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Regulation of Estradiol Valerate on Endometrial Receptivity in Mice with Embryonic Implantation Dysfunction

HUANG Xiao-qiong, HUANG Wei, SUN Xia, ZHONG Hai-chao, RAO Zi-liang, ZHONG Zhi-yong, HE Tian-tian, TIAN Ying-zhou, XU Min

2013, 33(1):  1-6.  DOI: 10.3969/j.issn.1674-5817.2013.01.001

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Objective To investigate the regulation of estradiol valerate on endometrial receptivity in mice with embryonic implantation dysfunction. Methods The female KM mice were randomized into negative control group,model group and estradiol valerate group ,with 33 mice for each group, to build embryo implantation dysfunction mouse model. Mice in estradiol valerate group were administrated by gastric perfusion of 0.2mg/kg estradiol valerate and mice in negative control group and model group was given by the same dose saline. Mice were sacrificed individually at 9∶00, 21:00 and 23∶00 in the 4th day after gestation, and uterus were taken out for detecting pinopode by scanning electron microscopy and the expression of MMP-9,TIMP-3,COX-2 and PGI₂ by immunohistochemistry. Results The pinopode of endometrium in negative control group was uniform distribution,significantly more prominent than processes on the surface of membrane,with almost same size and clear boundaries, which was the developing pinopode. The pinopode of endometrium in model group was reversible focal expression, with asynchronized development of pinopode, or the inconsistent size of pinopode. There were a large number processes in endometrium with inconsistent size, slightly lower than that in the negative control group:short and thin microvilli on the surface of processes also was the developing pinopode,slightly later development lagged behind than that in the negative control group on morphology. There was significant difference between the model group and negative control group by the expression of COX-2, PGI₂ and MMP-9 protein, protein of endometrial glandular epithelial cells and luminal epithelial cells (P<0.05), comparing with the negative control group, the expression of TIMP-3 protein in endometrial glandular epithelial cells and luminal epithelial cells had significant difference in the model group (P<0.01), valerate estradiol significantly increased the expression of COX-2, PGI2, TIMP-3 and MMP-9 protein in the endometrium (P<0.05). Conclusion Estradiol valerate could improve embryo implantation and it may works by indirectly promoting of pinopode formation in mice with embryonic implantation dysfunction, increasing of the expression of COX-2, PGI₂, TIMP-3 and MMP-9 in endometrium, and establishing endometrial receptivity, for improving embryo implantation dysfunction of mice.



Effect of Latrunculin A on Motility of Spermatozoa Cryopreservation in ICR Mouse

CHENG Fu-rong, YANG Shi-hua

2013, 33(1):  7-11.  DOI: 10.3969/j.issn.1674-5817.2013.01.002

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Objective To evaluate the effect of latrunculin A on motility of spermatozoa combined with optimized cooling rate of cryopreservation. Methods ICR mouse epididymal spermatozoa balanced in freezing medium were frozen at fast (-240℃/min), medium (-109℃/min), slow (-41℃/min) and super slow (-12℃/min) cooling rate for 5 min or 10 min respectively, before being plunged into liquid nitrogen for storage. Results No difference of sperm motility was observed between spermatozoa frozen for 5 and 10 min above liquid nitrogen at each cooling rate. Spermatozoa cryopreserved at super slow and medium cooling rate showed highest and lowest post-thaw motility respectively. ICR mouse spermatozoa were treated with 0.5 µmol/L, 1 µmol/L, 2 µmol/L and 4 µmol/L Latrunculin A before freezing and then cryopreserved at super slow cooling rate. The result showed that treating mouse spermatozoa with 0.5 µmol/L, 1 µmol/L and 2 µmol/L of Latrunculin A could not improve the motility of spermatozoa. However, high level (4 µmol/L) of Latrunculin A showed detrimental effect on ICR mouse sperm motility. Conclusion Cryopreservation proficiency of Latrunculin A on sperm is open to discussion.



Effect on Fertility by Active Immunization with Antigen Targgeting FSH Receptor Ligand Binding Region in Rats

SHI Yan, YU Lin, ZHU Yan, JIANG Ya-hong, SUN Zhao-gui, WANG Jian

2013, 33(1):  12-18.  DOI: 10.3969/j.issn.1674-5817.2013.01.003

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Objective To establish the rat model of premature ovarian failure by immune suppression to FSH receptor (FSHR), and to evaluate their phenotype. Methods On the basis of biological information of the whole FSHR protein structure, the extracellular domain of FSHR was cloned. The recombinant protein obtained by prokaryotic expression and three synthetic peptides of the protein were used to immunize 4 weeks old female SD rats. Estrous cycle, and blood FSH, estrogen and progesterone were detected separectely. Results After the active immunization, high titer antibodies were detected in the blood and lasted for 6 weeks. The antibodies were shown to specifically bind with the granulosa cells in vitro and at the same time, the ovarian response to gonadotropins was reduced in vivo. Estrous cycle disorders with shortened estrus, blood FSH rise and estrogen decrease were detected in rats immunized with recombinant protein; and the litter size was decreased while mating with normal males. Conclusion The active immunization in rats against FSHR ligand binding sites resulted in high titers of FSHR antibodies in the blood, which could specifically bind to ovarian granulosa cells, and resulted in lower response to gonadotropin in ovaries, and then symptoms of premature ovarian failure were observed in rats.



Construction and Preliminary Analysis of Subtractive Library for Cricetulus Barabensis with Albino Mutant

LI Ai-xue, ZENG Lin, SHANG Shi-chen, WANG Peng, YAO Xiao-lan, SUN Zhao-zeng

2013, 33(1):  19-22.  DOI: 10.3969/j.issn.1674-5817.2013.01.004

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Objective To construct the subtractive library for Cricetulus barabensis and its albino mutant and preliminarily analyse the obtained gene fragments that related to the hair mutation of Cricetulus barabensis. Methods The Cricetulus barabensis was used as tester and its albino mutant was used as driver, the subtractive Library was constructed by suppression subtractive hybridization (SSH) method. The positive clones was sequenced and analyzed. Results 126 Expressed sequence tags (ESTs) was obtained including 21 skeleton proteins, 42 transcription factors, 24 genes of metabolic enzymes, 25 transport factors and their regulators, and 4 genes of unknown. Not the common genes related to albinism but several kinds of genes related to the formation and endocytosis of lysosomes were found in the differentially expressed genes, suggesting that the production of albinism of Cricetulus barabensis may relate to the disfunction of transportation. Conclusion The subtractive library for Cricetulus barabensis and its albino mutant was successfully constructed and 126 ESTs was obtained. This study may lay a basis for looking for the genes related to albinism of Cricetulus barabensis and revealing the corresponding mechanism.



Cloning and Analyzing the Sequence of Resistin Gene in Macaca mulatta

LU Shuai-yao, WANG Jun-bin, YANG Feng-mei, YU Wen-hai, ZHAO Yuan, CHEN Li-xiong, LI Yan-yan, HE Zhan-long

2013, 33(1):  23-27.  DOI: 10.3969/j.issn.1674-5817.2013.01.005

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Objective To clone resistin gene of Macaca mulatta, its sequence has been identified and analyzed. Methods Total RNA was extracted from abdominal adipose tissue, and resistin gene was amplified by RT-PCR, cloned and inserted into vector. Sequence was identified by PCR and restriction enzyme digestion, and analyzed by DNAMAN software. The coding region was determined and compared by ORFfinder and BLAST software. Result Resist in gene (363 bp) was obtained by RT-PCR amplification and the recombinant cloning vector were positive identification by PCR and XbaI and PstI restriction enzyme digestion. Sequence analysis showed that the resistin gene homology was 96%, 67% and 65.7% with human, rat and mouse. Phylogenetic tree results showed that resistin gene of Macaca malutta has a close genetic relationship with human. The analysis results shows the resistin was coded to 108 amino acids and the homology was 92% with human. Conclusion The resistin gene of Macaca malutta was successfully cloned for laying a preliminary basis and providing experimental data in the related studies of resistin gene of Macaca malutta.



Establishment of EGFP Transgenic Rat

CHI Jun, ZHAO Ying, XUE Yi-jun, WANG Jin-jin, SUN Rui-lin, WANG Zhu-gang, FEI Jian, CHEN Guo-qiang, KUANG Ying

2013, 33(1):  28-32.  DOI: 10.3969/j.issn.1674-5817.2013.01.006

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Objective To establish an enhanced green fluorescent protein (EGFP) transgenic rat model, which will be used for organ transplant and gene therapy. Methods The lentiviruses carrying the EGFP driven by the CMV (cytomegalovirus) promoter were generated and injected into the perivitelline spaceof single-cell rat embryos obtained from superovulated Sprague Dawley females mated on the previous evening with Sprague Dawley males. The injected zygotes were transferred into oviduct of pseudopregnant SD females. Transgenic rat were identified by PCR analysis on genomic DNA from rat tail, the transcription and translation pattern of EGFP gene were detected by Real Time-PCR, Western-blot and fluorescent observation. Results After microinjection 210 injected zygotes were transferred. A line of EGFP transgenic rat was established. The green fluorescent protein was expressed in most of the tissues from the transgenic rat line, such as skin, muscle, pancreas, kidney, heart, testis, and so on. Conclusion The transgenic rat technical platform is established.



Comparative Observation on Three Kinds of Cranio-Orbital Injury Models in Rats

GAO Chen, ZHOU Hai-yan, LI Zhi-yun, ZHANG Jing-ke, JIA Pin, WANG Xiang-yang

2013, 33(1):  33-38.  DOI: 10.3969/j.issn.1674-5817.2013.01.007

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Objective To establish cranio-orbital injury models in rats which may help the research of the mechanisms and treatment of the injury. Methods Sixty rats were randomly divided into 3 groups, group A(forceps injury group),group B(free fall impact injury group) and group C(fluid percussion injury group). The cranio-orbital injury models on the left eyes of rats in the 3 groups were established respectively. The incidence of Marcus-gun pupil, exophthalmos, hypophasis, subhyaloid hemorrhage, optic nerve transaction, infection, orbit fracture, optic nerve sheaths hemorrhage, contusion and laceration of the brain in the model eyes and the mortality were counted respectively after surgery. The retina of normal eyes and model eyes were observed by microscope, the ultrastructure of retinal ganglion cells (RGCs) were observed under electron microscope. Results The performance of group A was superior to that of the other two groups with the standard of cranio-orbital injury models. The retina structure of model eyes was disorganized and the quantity of RGCs significantly reduced after injury. The ultrastructure of RGCs in model eyes showed the typical characteristics of apoptosis. Conclusion The animal models of cranio-orbital injury which according with the clinical reality can be established successfully with fluid percussion brain injury device (FPI) .This model provides a solid experimental foundation for future searching the mechanisms and treatment of the injury.



Establishment of Interleukin 1β-induced Recurrence Model in Rat with Acetic Acid-induced Gastric Ulcer cured by Omeprazole

JIANG Guo-rong, LIANG Guo-qiang, GE Hui-nan, WANG Chun-xiang

2013, 33(1):  39-43.  DOI: 10.3969/j.issn.1674-5817.2013.01.008

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Objective To establish the IL-1β-induced recurrence model in rat with acetic acid-induced gastric ulcer and cured by Omeprazole. Methods 1) The acetic acid-induced gastric ulcer rat model was reproduced by topical application of acetic acid, an improved method of Okabe. 2) Then, the rats with gastric ulcer were divided into 2 groups randomly, the rats of control group (spontaneous closure group) were intragastric administrated with physiological saline, while the rats of therapy group were administrated with Omeprazole. The rats Randomly sampled on the 14th, 21th and 28th day after treatment, together with the rats on the 28th day in control group, were executed at one time. Then the care rates(%), ulcer index, and the histopathological changes of gastric mucosa were assessed. 3) The cured rats were randomly divided into 2 groups, and were intraperitoneally injected with IL-1β of 0.75 mg/kg and 1.00 mg/kg respectively. The recurrence rate of ulcer (%), ulcer index, and histopathological changes of gastric mucosa were assessed 48h later. Results 1) The model rats induced by acetic acid showed typical ulcer when dissected randomly for observation. 2) After 14, 21 and 28 days’ administration of Omeprazole, the cure rates were 25.0%, 37.50% and 100.0%, respectively. The thickness of regenerative gastric mucosa increased with the extension of therapy time. While the number of cystic-dilated glands, inflammatory cells and capillary vessels reduced as the treatment time increased. The cure rate of spontaneous closure with 28 days was 37.50%. 3) In the IL-1β-1.00 mg/kg group, the recurrence rate of ulcer was 100%, and the ulcer index was 18.0±3.4. Moreover, compared with rats 28 days after treatment, the regenerative gastric mucosa was significantly thinned, but the number of cystic-dilated glands, inflammatory cells and capillary vessels obviously increased. Conclusion The model of interleukin1β-induced recrudescence model in rat with acetic acid-induced gastric ulcer which has been treated by Omeprazole was applicable to the study of prevention and treatment of recurrence of peptic ulcer.



Establishment of Gastric Bypass Model and Its Impact on Weight Loss in Rats

WANG Yi-long, Jiang Ming-zhe, Cai Jing-li, Chen Deng-gang, Xie Jie-bin, Li Li-jun, Liu Chang-bao, Chen Rong

2013, 33(1):  44-46.  DOI: 10.3969/j.issn.1674-5817.2013.01.009

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Objective To establish the model of gastric bypass surgery (RYGB) in SD rats, and to study the effect of gastric bypass surgery on weight loss. Methods Thirty five male SD rats were randomly divided into three groupsłşgastric bypass surgery was performed in 15 male rats as the study group (O group), sham operation was performed in 10 male rats which were given diet control after operation as the study control group (C group), and laparotomy was performed in 10 male rats which were given normal diet control after operation as the blank control group (B group). Body weight and food intake were recorded before and after surgery. Results The number of death rats was 7 in the study group, and the survival rate is 53.3%; the maximum weight loss was happened in the first week after surgery, thereafter weight rebounded slower than group B and C (P<0.05). Conclusions It is feasible to established gastric bypass surgery model in rats. RYGB can control weight of rat effectively.

Antitumor Activity of Histone Deacetylase Inhibitor in Osteosarcoma Cells in vivo

CHENG Dong-dong, LIU Lei, ZHANG Zhi-chang, YANG Qing-cheng

2013, 33(1):  47-51.  DOI: 10.3969/j.issn.1674-5817.2013.01.010

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Objective To establish an animal model of human osteosarcoma in nude mice, and use the model to investigate the antitumor activity of Trichostatin A with this animal model. Methods Nude mice received MNNG/HOS cell suspensions was subcutaneously injected into nude mice with the concentration of 2×10⁶ to develop growing tumor. Tumor-bearing mice were examined by Micro-CT imaging system; the tumor tissue was observed by microscopic examination after HE staining. The animal model of human osteosarcoma was established. When subcutaneous tumors were 10-20 mm, mice were randomly divided into the control group (n=6), TSA group (n=6) and CTX group (n=6), the TSA group was treated with TSA (1 mg/kg); CTX group was treated with CTX (20 mg/kg); and control animals were treated with vehicle containing saline with 5% DMSO. Mice were injected every three days. Measure the size of tumor and the weight of tumor-bearing mice was recorded. The tumor growth curve and nude mice weight change curve were drawn. Results Two weeks after the inoculation, local tumor was formed. Four weeks later, bone destruction was visible in Micro-CT imaging system; osteoid tissue formed by osteosarcoma cells could be seen under light microscope and osteoplastic process was ubiquitous. TSA significantly inhibited tumor growth (P<0.05). No significant body weight difference or signs of overt toxicity was noticed between TSA and control group. Conclusion The animal model of osteosarcoma was successfully established, which will provided the basis of animal experiments for clinical studies of osteosarcoma. TSA significantly inhibited tumor growth of osteosarcoma in vivo.



Effect of Recombinant Growth Hormone on Nutritional Status and Pulmonary Function in COPD Rat

WEN Wen, LIU De-ling, GUAN Ze-jin, HUANG Yu-hai, CHEN Mei-lian, YE Jia, LAI Guo-xiang

2013, 33(1):  52-55.  DOI: 10.3969/j.issn.1674-5817.2013.01.011

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Objective To study the effect of gene recombinant growth hormone (GH) on nutritional status and lung function of chronic obstructive pulmonary disease (COPD) rats. Methods The COPD rats was established through smoking combined with lipopolysaccharide, 16 cases of COPD rats were randomly divided into 2 groups, group GH for growth hormone treatment (8 cases), daily subcutaneous injections of recombinant rat growth hormone 1 µg,7 days of continuous use, group NS (8 cases) with subcutaneous injection of physiological saline. Fifteenth days after treatment, the rat body weight, serum albumin (ALB), transferrin (TRF), prealbumin (PA), creatinine, blood urea nitrogen and glucose of all the rats were detected, and the lung function were evaluated. Results The COPD model conforms to the COPD basic pathological changes. In two groups before treatment weight, albumin, prealbumin, total protein, creatinine, blood urea nitrogen, blood glucose had no significant difference (P>0.05), body weight, albumin, prealbumin, total protein of growth hormone group increase has higher than the control group (P<0.05), creatinine, urea nitrogen were significantly decreased (P<0.05), the glucose increased before and after treatment with growth hormone group, but the difference was not statistically significant (P>0.05). Lung function (FEV0.3, FEV0.3/FVC, PEF) after treatment compared with the saline control group were significantly higher (P<0.05). Conclusions The growth hormone therapy can improve nutritional status and lung function of the stable phase of chronic obstructive pulmonary disease rat.



Assessment of Thyroid Function on High-fat Diet Insulin Resistant Rats

ZHANG Ying-yan, YANG Jing, DU Jian, AO Na, Liu Yu-ling, Wang Tian-bao

2013, 33(1):  56-59.  DOI: 10.3969/j.issn.1674-5817.2013.01.012

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Objective To assess the thyroid function on the high-fat insulin resistant rats. Methods Insulin resistant rats were induced by high-fat diet feeding,then assessed by euglycemic-hyperinsulinemia clamp technique. Determine the index of thyroid function and serum lipid. Results (1) The glucose infusion rate(GIR_60~120) was significantly decreased in HF group, (P<0.05). The level of serum TC、TG、LDL-C、FFA were significantly increased while HDL-C was decreased in high-fat diet (HF group), P<0.05. (2) The differences of TSHŁ¬FT3and FT4between the NC and HF group were not statistically significant (P>0.05). The serum level of TSH has a positive correlation with TG (R=0.749, P<0.05) and has a negative correlation with HDL-C (R =-0.172, P<0.05). There were uncorrelation between TSH and TC, LDL-C, FFA. Conclusion The thyroid function of the high-fat diet insulin resistant rats was no significant differences with the normal rats,but the serum TSH was close to the serum level of lipid.

The Expression of Survivin mRNA on Thymus Tissue Apoptosis Induced Radiation

QU Jing -kai, WANG Hong-yan, QI Ya-li, CHEN Yu-bing, SONG Xiang-fu, GONG Shou-liang

2013, 33(1):  60-63.  DOI: 10.3969/j.issn.1674-5817.2013.01.013

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Objective To observe the expression of survivin mRNA on thymus tissue apoptosis induced by ionizing radiation and study its role in radioactive thymus tissue apoptosis. Methods The mouse were irradiated with the X rays (1.75 Gy per time, 1 time per week for consecutive four times, total doses of 7 Gy).The mouse were killed and the thymus tissue were taken out at 30 and 60 d after they were irradiated(six mouse per time). The cell cycle and apoptosis rate were detected by flow cytometry. Survivin gene mRNA expression was detected by RT-PCR. Results The thymocytes and the of cells in G0/G1 and S phase in irradiation group were similar to that in control group, but the G2/M phase cells percentage was lower after irradiated 30 d and higher after 60 d compared with the control group (P<0.05).The thymocyte apoptosis in irradiation group was higher than that in control and the expression of survivin mRNA reduced significantly (P<0.05) at respectively time point. Conclusion Ionizing radiation can promote apoptosis in thymus by reducing survivin mRNA expressions.



The Comparative Studies on Percutaneous Absorption for Two Kind of Terbinafine Hydrochloride Cream by in vitro Tibet Minipig’s Skin

LUO Rong, ZHOU Lin, CHEN Wei

2013, 33(1):  64-68.  DOI: 10.3969/j.issn.1674-5817.2013.01.014

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Objective To study the percutaneous absorption of terbinafine hydrochloride cream through in vitro Tibet minipig’s skins, and compare the penetration absorption of domestic certain enterprise product with GlaxoSmithKline china product. Methods Using the modified Franz diffusion cell, samples were collected at different time after the drug was applied. The concentration of terbinafine hydrochloride in the samples was measured by HPLC. Results The penetration release quantities of terbinafine hydrochloride across Tibet minipig skins in 80% ethanol saline solution in 48h were very low, only 63.33 µg·cm^-2 for GlaxoSmithKline China product, and 55.23 µg·cm^{- 2} for certain enterprise product, and the percolation rate were 1.19% and 1.09%, respectively. Conclusion The percutaneous absorption of terbinafine hydrochloride cream was very low between two products without significant difference. The penetration absorbency through Tibet minipig skins of certain enterprise cream is similar to GlaxoSmithKline china product. The most of the main components of terbinafine hydrochloride cream remain in the surface of the skins, only a few through the skin barrier. It was more suited to local cure.

Research Advance on Parkinson’s Disease Cell Model

FAN Zhen-qun, LIU Shu-min, ZHOU Shi-hui, WANG Ke-xin

2013, 33(1):  74-78.  DOI: 10.3969/j.issn.1674-5817.2013.01.017

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Parkinson’s disease, which serious endangers the health of middle- aged and elderly pepole, has attracted more and more attention in these years. In the study of Parkinson‘s disease, the in vivo experiments will spend more time and money. Because of that, a lot of in vitro models have been used to reflect the pathological changes and the efficacy of new drug. Many reagents have their own specialities, and different cell lines also have their own characters, and their cell model will finally influenced the research outcome.