Table of Content

25 April 2013, Volume 33 Issue 2

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Effects and Preliminary Mechanisms for Diethylstilbestrol on Follicular Development in Prepubertal Mice and Rats

GU Yan-qiong, SHI Yan, WANG Jian, SUN Zhao-gui

2013, 33(2):  79-83.  DOI: 10.3969/j.issn.1674-5817.2013.02.001

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Objective To observe the effects of the synthetic non-steroidal compound diethylstilbestrol on the ovarian folliclar development in the prepubertal mice and rats, and the involved mechanisms in the follicular synchronization resulted from the diethylstilbestrol treatment. Methods The prepubertal mice and rats were treated by intraperitoneal injection of a certain dose of diethylstilbestrol, and then the ovaries were collected to count the number of tertiary follicles in the ovary by serial sectioning and HE staining. On the other hand, the immunohistochemistry and immunoblotting methods were used to detect the distribution and quantity of Ubiquitin Carboxyl Terminal Hydrolases L1 (UCH-L1), c-Jun activation domain binding protein1 (Jab1), and cyclin dependent kinase inhibitive factor (p27^Kip1) in the ovaries, separately. Results Immunohistochemical results showed that the localization of UCH-L1 was only restricted into oocytes, Jab1 was detected in the oocyte nuclei of secondary follicles and tertiary follicles, and p27^Kip1 was distributed extensively in the ovary. Only considering the oocyte, in response to DES treatment, UCH-L1 and Jab1 staining signals were enhanced, and p27^Kip1 signal was become weak in the nucleus, similar to the situation in the tumor cells. However, the protein levels of all UCH-L1, Jab1 and p27^Kip1 in ovaries were showed increasing tendency in the DES-treated group to some degrees compared with naive group. In similarly rising tendency, the protein levels of UCH-L1 and p27^Kip1 were changed with the age in the ovaries of 1-4 week old mice though Jab1 was found no obvious change. Conclusion Intraperitoneal injection of diethylstilbestrol caused ovarian follicular growth synchronization, and involved mechanisms may be that UCH-L1 and its associated protein Jab1 down-regulated p27^Kip1 in the oocytes nucleus during the diethylstilbestrol-induced follicular growth synchronization.



Analysis on Differential Genes Expression in Early Fetuses Derived from in vitro Fertilization Using Oocytes from Induced Diabetes Mouse

TAO Ling-yun, QIANG Su-jing, GAO Cheng, HU Jian-hua

2013, 33(2):  84-89.  DOI: 10.3969/j.issn.1674-5817.2013.02.002

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Objective To study the effect of maternal diabetes environment on early fetuses gene expression through in vitro Fertilization(IVF) technology. Methods Diabetes animal model was induced by a low dose of STZ in 6-8 weeks ICR female mice, and then the mice with hyperglycemia were superovulated. Two-cell embryos derived from in vitro fertilization were transferred into the oviduct of normal pseudopregnant ICR female mouse. Total RNA of 14 days’ fetuses were collected and detected by Agilent 2100 Bioanalyzer system. Through subsequent reversed transcription, label, hybridization, elution and microarray scann by Affymetrix Scanner 3000, finally raw data was read by Command Console Software 3.1. The experiment was repeated three times. Results 121 differentially expressed genes (P<0.05) were screened out. The expression level of 119 genes in the experimental group was less 0.5 times than that in the control group, and it shown more than 2-fold up regulation. Conclusion Maternal diabetes environment influenced the growth and development of the oocytes through up-regulating the metabolism-related genes and down-regulated development-relating genes and further influenced the growth and development of the early embryos.



Preliminary Research in Establishment, Characterization, and Application of Human Primary Esophageal Cancer Xenograft Models

OU-YANG Ke-dong, LIU Ji-bin, WANG Ke, HU Gang, GU Ying, XIE Fu-bo, ZHAO Qiang, ZHANG Ya-hua, YANG Wei-min, WENG Dan-yi, ZHANG Yi-xin, QIN Xiao-ran

2013, 33(2):  90-98.  DOI: 10.3969/j.issn.1674-5817.2013.02.003

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Objective To establish and characterize human primary esophageal xenografts by subcutaneous implantation of fresh resected esophageal cancer (EC) specimens in immunodeficient mice with unchanged tumor histological features and validate their application value. Methods Thirty-six fresh resected human esophageal cancer specimens were subcutaneously implanted into SCID mice. After tumor developed, they were serially passaged with nude mice using the same methods. For each case, when the xenograft tumors reached size 500-1000mm³, tumors were excised and devided into four parts: the first part was implanted into nude mice for the growth of next generation; the second part of tumor was viably saved in liquid nitrogen for future model recovery; the third part of tumor was snap frozen for DNA/RNA extraction; and the last part of tumor was fixed in formalin and embedded into paraffin (FFPE) for H&E staining and pathology analysis. Using four cases of human primary EC xenografts passaged in nude mice, chemosensitivity study in vitro and efficacy study in vivo were performed to evaluate the application value of these models. Results Twenty-three in 36 cases by fresh EC specimens implantation were successfully developed into tumor in immunodeficient mice, the implantation success rate was 63.89%. These xenografts were grown up stably and could be continuous passaged to next generation. Histopathology examination of transplanted tumors showed that these xenografts were stable with similar histological features compared with the original donors. Twenty-one in 23 models were successfully established in SCID mice when implanted with recovering cryopreserved xenograft tissues, the recovery success rate was 91.30%. In in vitro chemo-sensitivity assay, the selected four cases were sensitive to Sorafenib, 5-FU, Doxorubicin, Tarceva and Irinotecan; however, there were individual differences in sensitivity to Docetaxel, even more, ESX003 and ESX004 were showed no response to Docetaxel. At dosage of 100 mg/kg, Irinotecan could significantly inhibit the growth of these four human primary EC xenografts in nude mice with relative tumor proliferation rate (T/C) of 22.02%, 39.63%, 21.69% and 39.70%, respectively. Conclusions Human primary EC xenograft models could be established with holding original donor’s histological features. The cryopreserved xenografts tissues could be recovered successfully in SCID mice, indicating that these xenograft models can be used repeatedly. The results in chemosensitivity study in vitro and efficacy study in vivo with four selected EC xenograft models show that the human primary EC xenograft models are valuable and very helpful for anti-cancer drugs screening and translational research of human esophageal cancer.



Establishment of Colon Cancer Mouse Model and its Biological Characteristics

WAN Bo-shun, YU Jing-xian, CHEN Yue-yu, WANG Xiao-min, ZHAO Fang-yu, YAO Ming, YAN Ming-xia

2013, 33(2):  99-105.  DOI: 10.3969/j.issn.1674-5817.2013.02.004

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Objective To establish subcutaneous and orthotopic transplant mice models by using human colon cancer clinic sample, in order to provide a useful tool for studying the characteristics of colon cancer and preventing and curing the colon cancer. Method Fresh colon cancer sample was transplanted subcutaneously into BNX mice and nude mice, then the tumor xenografts were maintained by serial passages in BNX mice and nude mice in order to establish subcutaneous transplantable tumor animal model, tumor size was monitored regularly every week. The tumor tissues were collected and orthotopically implanted into the cecum sub-serosa. The rates of tumor formation and metastasis were observed after animal sacrifice. The expression of ESA、CEA、C-erbB-2、P53 proteins were detected by immunohistochemistry. DNA aneuploid and cell cycle distributions of tumor cells were estimated by flow cytometry. Result The subcutaneous transplant model of colon cancer was established, the tumor formation rate of orthotopic transplant model was 62.5% without metastasis. Biopsy observation indicated that the xenograft tumor was pathologic stage II adenocarcinoma as same as the original human carcinoma. The expressions of ESA and CEA were strongly positive, C-erbB-2 and P53 were positive by immunohistochemistry. Flow cytometry was used to detect the change of cell cycle, the results indicated that the ratio in G₀ and G₁-phase was 67.50%±5.59%, 4.47%±2.31% in G₂ and M-phase and 28.02%±7.13% in S phase. DNA heteroploid detection showed that the heteroploid ratio of xenograft tumor was 1.74±0.02. Conclusion The subcutaneous and orthotopic transplant colon cancer model in mice was successfully established, which had the same biological characteristic as original human colon cancer.



Chronic Hypoxia- high Carbon Dioxide -induced Cerebellum Edema and Protective Effect of Curcumin in Rat

MAO Xiang-lan, ZHAO Xiang, LI Ze-jie, ZHANG Tao, CAI Hong-yong, YE Guang-hua, YU Lin-sheng

2013, 33(2):  106-111.  DOI: 10.3969/j.issn.1674-5817.2013.02.005

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Objective To explore expression of AQP4 and edema in hypoxia- high carbon dioxide rat cerebellum edema model and the protective effects of curcumin. Methods Fifty-five adult male SD rats were randomly divided into 3 groups: the control group, the experimental group and curcumin treated group. The experimental group and curcumin group were housed in atmospheric hypoxia- high carbon dioxide environment to induce hypoxia-high carbon dioxide brain edema model. The difference of the hypoxia-high carbon dioxide of rat cerebellar cells edema degree were observed under electron microscopic after HE staining and immunohistochemistry. The expression level of AQP4mRNA in the experimental group and the control group were detected using Real time fluorescence PCR. The results showed that cerebellar cells edema was more significant and AQP4 gene expression was relatively higher in the experimental group rat than curcumin group. Conclusion Rat cerebellar edema can be developed in hypoxia-high carbon dioxide environment, and it may be related to the expression of AQP4 gene, Curcumin has protective effect on such edema.



Structure and Composition of Intestinal Mucosal Barrier in Zebrafish (Brachydanio rerio)

ZHANG Lin-li, YAO Yi-lin, CHU Xiao-hong, ZHANG Qian, YANG Ping, BIAN Xun-guang, LIU Yi, HU Jian-hua, GAO Cheng, LIN Jin-xing, CHEN Qiu-sheng

2013, 33(2):  112-116.  DOI: 10.3969/j.issn.1674-5817.2013.02.006

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Objective To observe and analyze the structure and composition of intestinal mucosal barrier in zebrafish (Brachydanio rerio). Methods The morphology was observed by transmission electron microscopy, histochemistry and immunohistochemistry were undertaken on the organizational structure and cell composition of intestinal mucosal barrier in the zebrafish. Results The zebrafish intestinal absorptive cells had developed junctional complex, top-down followed by visible tight junctions, intermediate connections, and desmosomes. They form a closed mechanical barrier. The intestinal mucosa absorption cell distributed between rich goblet cells, especially in the largest number of the intestine. PAS reaction was significant positive. The goblet cells were the chemical major cells for the intestinal mucosa barrier, whose secretions in the intestinal cavity surface to form a layer of mucus layer. Their intestinal intraepithelial lymphocytes and mast cells were the intestinal mucosa of primary immune cells. The mucosal epithelium of endocrine cells in both open and closed, striated border developed in a linear (or membrane) in the intestinal epithelium lining the cavity surface. Conclusions The intestinal mucosa barrier system of zebrafish consisted of relative complete mechanical barrier, chemical barrier and immune barrier structure. So zebrafish could be used as animal models for the study of intestinal mucosal barrier.



Transcranial Injection of Ethanol for Establishing Brain Injury Model in Rat

HE Ji-gang, SHEN Zhen-ya, TENG Xiao-mei, DING Ying-long

2013, 33(2):  117-120.  DOI: 10.3969/j.issn.1674-5817.2013.02.007

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Objective To establish a stable rat model of brain injury. Methods Ten Rats were transcranially injected with ethanol. Results The success rate of rat brain injury model is 100%, and 10 sham-operated rats survived too. After four weeks experimental group and sham group was no significant difference in body weight and daily food intake. (376.1±11.5 g vs 353.2±10.3 g; P=1.534); (23.4±3.1 g vs 19.8±2.3 g; P=0.723). By brain magnetic resonance imaging, pathology HE staining, and brain tissue slices TTC staining can confirm modeling success. Conclusion Transcranial injection of ethanol to establish rat brain injury model is simple. Success rate is higher than traditional methods. A solid foundation is established for further studies on brain injury.



Effects of Hypoxia on Proliferation of Hippocampal Neural Stem Cells in Rat

WANG Shi-qiang, LOU Shu-jie

2013, 33(2):  121-125.  DOI: 10.3969/j.issn.1674-5817.2013.02.008

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Objective To observe the effects of hypoxia on proliferation of rat’s hippocampal neural stem cells. Methods The hippocampal neural stem cells from newborn SD rats were cultured in the concentration of 1%, 4%, and 20% oxygen respectively, and the diameter and number of neurospheres were observed under the low oxygen. Results (1) Effects of hypoxia on proliferation efficiency. The number of neurospheres of 1% group was significantly lower than control group, (P<0.05). The number of neurospheres of 4% group was higher than control group, but no significantly difference. (2) Effects of hypoxia on proliferation rate. The diameter of 1% group was significantly shorter than control group at 24 h, 36 h and 48h,and reached the extremely significant levelat 48 h (P<0.01). The diameter of 4% group was longer than control group in 24 h, 36 h and 48 h. There was a significant difference at 36h and an extremely significant difference at48 h. (P<0.01). Conclusion 4% oxygen concentration can not only improve proliferation efficiency of rat’s hippocampal neural stem cells, but also improve its proliferation rate. 1% oxygen concentration can not only inhibit proliferation efficiency of rat’s hippocampal neural stem cells, but also inhibit its proliferation rate. This suggests us that moderate hypoxia can improve the proliferation of rat’s hippocampal neural stem cells, while server hypoxia inhibits its proliferation.



Isolation and Purification of Adult Tibet Minipig Pancreas Islets in vitro

QI Wen-jun, WANG Yi-nan, HU Qiang, TANG Hua, NASHUN Ba-ya-er, GU Wei-wang

2013, 33(2):  126-129.  DOI: 10.3969/j.issn.1674-5817.2013.02.009

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Objectives Identify the optimized techniques for the isolation and purification of pancreas islets to supply high quality islets for xenotransplantation in the treatment of diabetes. Methods Compare the quantities and effects of different mass concentration (1 mg/ml, 2 mg/ml, 3 mg/ml, 4 mg/ml) and the same mass concentration conditions at different time points (0 min, 15 min, 30 min, 45 min, 60 min, 75 min, 90 min) on Tibet minipig pancreas islets isolation. Compare the quantities and effects of Ficoll400 density gradient centrifugation, lymphatic separation liquid centrifugal, centrifugal, and placing the 4 ways on purification of islets. With AO/PI dyeing calculation islet cell survival rate. Results The best time points of different mass concentration collagen enzyme to acquire islets were confirmed, 1 mg/ml and 2 mg/ml groups for 45 min,3 mg/ml and 4 mg/ml groups for 30 min respectively. Among the 4 best time points, 1 mg/ml group gets the most amount of islets . Each group gets islets of complete morphology at best time point respectively. Islets number by many to less is placing> Ficoll400 density gradient centrifugation>lymphatic separation liquid centrifugal>centrifugal among 4 kinds of purification ways. The AO/PI staining after testing the survival rate of pancreatic islet cells for more than 95.00%. Conclusions In the condition of cutting tissue into pieces, collagen enzyme mass concentration 1 mg/ml, 45 min of digesting time can get more islets, Ficoll400 density gradient centrifugation collects islets most effectively.



The Cytotoxicity Test and Rejection Observation of Abdominal Zipper for Miniature Pigs

YU Chen-lin, TANG Qing, LIU Zhi-xue, MA Lei, XU Chen, CAI Li-ping, CUI Shu-fang

2013, 33(2):  130-134.  DOI: 10.3969/j.issn.1674-5817.2013.02.010

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Objective To assess the safety of abdominal zippers of miniature pig by cytotoxicity test and rejection observation. Methods The safety of abdominal zippers of miniature pig were evaluated by CCK-8 cytotoxicity test. In the test of rejection observation, nine miniature pigs were randomly divided into three groups: control group(C group), surgical procedure group(S group) and the animal model of miniature pig with abdominal zipper group(M group), and IL-1, IL-6, IL-10 and TNF-〈 in blood serum were detected before surgery (0 d), and during the day of 1d, 3 d, 7 d, 14 d and 21 d after surgery. Results The cytotoxicity grade was 0 orⅠlevel, and the level of IL-1, IL-6, IL-10 and TNF-〈 in S group and M group increased significantly at 1d and 3 d after surgery, but there was no difference between S group, M group and C group at 7 d, 14 d and 21 d after surgery. Conclusions The abdominal zipper for miniature pigs, which satisfies the national safety standard of biological materials, has qualified cytotoxicity and excellent biosafety.

Changes of Biochemical Indices and Tibia Morphology of Stress Fracture in Rabbits

ZHAO Xian-zhe, ZENG Gui-gang, QIAO Wei-wei, ZHANG Shen

2013, 33(2):  135-138.  DOI: 10.3969/j.issn.1674-5817.2013.02.011

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Objective To investigate the changes of the content of calcium, phosphorus and zinc in serum and the morphology of skeleton in the development of stress fracture in rabbits. Methods Sixty male rabbits were randomly divided into control group(n=30) and experimental group(n=30). Let the experiment rabbits run and jump for 300 times per day with high voltage and under-current stimulation, 6 times a week ,and totally 5 weeks.Six rabbits each group were sacrificed every week for the determination of the content of calcium, phosphorus and zinc in serum and tissue, for determination of the content of parathyroid hormone, osteocalcin and calcitonin in serum, and for observing the pathological changes of tibia. Results The injury of tibia tissue was exacerbated as training time increased. The serum zinc from experimental group was significantly higher than that of the control group(P<0.05) after 3 weeks training. The content of parathyroid hormone, osteocalcin and calcitonin in serum showed no difference between experimental and control group. Conclusions The element of calcium and zinc might play some roles in the development of the experimental stress fracture.



Introduction of a Micro-Surgery Model through Femoral Arterial Catheters and Gastric Cannula in Mice

YUE Xia, Ruth Magaye, WANG Zun, DAI Na, ZHAO Jin-shun, ZOU Bao-bo

2013, 33(2):  139-143.  DOI: 10.3969/j.issn.1674-5817.2013.02.012

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Objective A micro-surgery model through femoral arterial and gastric cannula in mice is introduced. Methods After mice were anesthetized by inhalation of isoflurane, catheters were placed into the femoral artery. Thereafter, gastric cannula was tunneled subcutaneously through two incisions between the back of the neck and the left upper quadrant. After cutting through the abdominal muscle, the stomach was exposed. Gastric cannula was then implanted into the lower end of the stomach through an incision on the gastric wall. Heparin (20 u,12~15 µl/h) were continuously infused in both catheters through a double-channel swivel connected with a pump. Result All mice fully recovered after surgeryŁ¨1ˇ«2 daysŁ©. The artery catheters and gastric cannula work well until 7 day after micro-surgery operation. Wound infection, gastric fistula and peritoneal abscess were not found. Conclusion This is a dynamic, safe, as well as efficient micro-surgery model, with less injury to the mice and smaller foreign matter in the mouse stomach. It will be a useful model for pharmacological, toxicological, and clinical experimental studies.



Effects on Hematology and Serum Chemistry Parameters after Single or Multiple Blood Collection from Tail Vein of Rats

LIU Yong-zhen, WANG Hao, DU Ming, WU Jun-liang, GONG Li-kun, REN Jin

2013, 33(2):  144-148.  DOI: 10.3969/j.issn.1674-5817.2013.02.013

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Objective To evaluate the effects on hematology and serum chemistry parameters after single or multiple blood collection from the tail vein of rats, and to investigate whether blood collection for toxicokinetic analysis can be performed on the same animals from the main study rats or recovery rats in toxicity studies. Methods Fifty Sprague Dawley rats were randomly divided into 5 groups (5 per gender per group). Two models were used in this study. In Model 1 (imitating a single dose toxicity study), there are two groups including no blood sampling group and multiple blood sampling group. In Model 2 (imitating a 28-day toxicity study), there are three groups including no blood sampling group, single blood sampling group and multiple blood sampling group. Blood was collected via an abdominal artery to evaluate the effects on hematology and serum chemistry parameters on Day 2 (Model 1) and Day 29 (Model 2) respectively. Results Hematology and serum chemistry parameters were not affected in the male rats in Model 1 after multiple blood sampling and in both male and female rats in Model 2 after single blood sampling. However, erythrocyte parameters were affected in the female rats in Model 1 and in both genders in Model 2 after multiple blood sampling. Changes in several serum parameters were also noted in the rats in Model 2 after multiple blood sampling. Conclusion In rat toxicity studies, there is a possibility to perform multiple toxicokinetic blood sampling on the main study rats or on the recovery rats if the total blood loss can be controlled within a certain time interval.

Progress in Preparation Methodology of Animal Models of Burn Injuries

LIU Qin, DIAO Bo, ZHANG Ying

2013, 33(2):  162-166.  DOI: 10.3969/j.issn.1674-5817.2013.02.018

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Currently, the changes of the body system and the local wounds after burn injuries are not entirely clear, which results a series of problems in burn treatment. The animal models of burn injuries are important tools for researching the burn treatment. In this paper, the methods of preparation of animal models of burn injuries were reviewed, in order to provide some references for the improvement and development of preparation methodology of animal models of burn injuries.