No Evidence of Effect on Male Mice Germ Cells After Acute Treatment With Thiram

Thiram is a dithiocarbamate compound widely used for industrial processes and agriculture. Animal studies reveal that this compound may afftct the male reproductive system. Aim of this study was to test, using sensitive testicular parameters, whether thiram directly affects germinal cells. For this purpose, B6C3F1 mice were intraperitoneally injected with thiram in oil (single dose:75 mg/kg; repeated five daily doses: 25 mg/kg).Although both treatments were toxic, none of the parameters examined, i.e., testis weighi, spermatid head number,specific enzyme levels at different times after treatment (14, 28, 35, 56 days) showed significant variations from the controls, On the contrary, in the positive controls (treated with chlorambucil), a marked reduction of sperm head number as well as a deerease of lactate dehydrogenasex and sorbitol dehydrogenase activity letels were evidenced at day 28, with a tendency to recover at day 35. Under these conditions thiram did not cause cytotoxicity on differentiating spermatogonia and on late spermatocyte stages of mice gonads

Antitumor effects of artesunate on human breast carcinoma MCF-7 cells and IGF-IR expression in nude mice xenografts

Purpose： The objective of this study was to investigate the anti-tumor effects and analyze the mechanism of artesunate （ART） action on breast cancer in vivo using tumor transplanted nude mice. Methods： The human breast tumor cell line MCF-7 was transplanted into nude mice, and the animals were treated with various doses of ART alone or in combination with cyclophosphamide （CTX） or normal saline （NS）. The tumor inhibitory effects were observed and compared, and the ultrastructural morphology of the transplanted tumor cells was observed by electron microscopy. The apoptosis rates and cell cycle status were detected by flow cytometry （FCM）. The expression of apoptosis-related proteins p53, Bcl-2, Bax and Caspase-3 were detected by immunohistochemistry and IGF-IR was detected by western blot. The expression correlation for these proteins was also analyzed. Results： The tumor inhibition rates in the low dose ART group, high dose ART group, CTX group and combined drug therapy group were （24.39±10.20）%, （40.24±7.02）%, （57.01±5.84）% and （68.29±5.1）%, respectively. The cell cycle was arrested in phase G0/Gt after treatment with ART. The expression of Bcl-2 was significantly reduced, and the expression levels of Bax and Caspase-3 were significantly increased in the ART group compared to the negative control saline group. There was no significant difference detected in p53 expression. The Bcl-2 level was negatively related to Bax and Caspase-3. The western blotting results showed IGF-IR downregulation. Conclusions： ART inhibits the growth of MCF-7 breast tumor cell xenografts in nude mice. The anti-tumor mechanism of ART for human breast carcinoma in nude mice might be correlated with the alteration of apoptosis related protein expression, which may further induce apoptosis and inhibit cell proliferation.

Carboxymethytl Pachymaram Up-Regulates Dendritic Cell's Function in Hepatitis B Virus Transgenic Mice in vitro

The effect ofcarboxymethytl pachymaram （ CMP ） on the function of dendritic cells（DCs） derived from spleens of hepatitis B virus transgenic mice are studied in vitro. The phenotypes of DCs are tested by flow cytometry （FCM）, cytokines measured by ELISA. The expression of DCs＇ phenotypes in IdBV transgenic mice are low （CD80^＋CD11c^＋：59.12±11.53 vs 9,60±4.53, p〈0.01; CD80^＋ MHC-Ⅱ^＋： 44.86±12.31 vs 9.80±5,72, p〈0.01, normal mice vs HBV transgenic mice）, the ability of DCs stimulating T lymphocytes proliferation decreases （0.37±0.11 vs 0.20±0,11, p〈0.05, normal mice vs HBV transgenic mice）, levels of IL-12 and IFN-y decrease whereas the level of IL-10 increases; CMP can enhance DCs＇ ability of stimulating T lymphocytes proliferation, facilitate the secretion of IL-12 and IFNp, inhibit the secretion of IL-10, thus up regulates DCs function. The results show a good prospective use of CMP on the treatment of chronic hepatitis B.

Optimal dose of busulfan for depleting testicular germ cells of recipient mice before spermatogonial transplantation

Successful spermatogonial transplantation requires depletion of the host germ cells to allow efficient colonization of the donor spermatogonial stem cells. Although a sterilizing drug,busulfan （Myleran）,is commonly used for preparing a recipient mouse before transplantation,the optimal dose of this drug has not yet been defined.The present study investigated the effects of different doses of busulfan （10-50 mg per kg body weight） on survival rate,testicular mass and histomorphology,and on the haploid spermatids and spermatozoa of male BALB/c mice.The results suggest that a dosage of 30 mg kg^-1 is optimal for the ablative treatment withbusulfan used to prepare the recipient mice. This dose results in an adequate depletion of the host germ cells for colonization of donorderived spermatogonial stem cells and causes the lowest death rate of the animals.

Effect of Quercetin on Breeding and Apoptosis of Cervical Cancer HeLa Cell and on Growth of Transplanted Tumor in Nude Mice

Effect of quercetin on HeLa cell system of cervical cancer was studied by methods of MTT and Annexin V-FITC/PI. The results show that quercetin has functions of inhibiting breeding of HeLa cells and inducing apoptosis of the cells. The total apoptosis rate is positively proportional to reaction duration and concentration of quercetin used. The maximum apoptosis rate being （88.76±2.35）% was obtained when the concentration was 50.0 μmol/L and the cells were treated with quercetin for 72 hours. Based on establishing a model of tumor of cervical cancer transplanted into nude mice, quercetin of different concentrations was injected into abdominal cavity of nude mice and situation of tumor growth was reviewed. The result showed that with quercetin concent＇ration increasing from 0 to 100.0 μmol/L, the transplantation volume and weight of the tumors decreased from （279.59±70.58） mm^3 and （0.145±0.019） g to （128.72±36.12） mm^3 and （0.089± 0.019） g respectively, while apoptosis rate of the transplanted tumor increased from （9.63±1.85）% to （34,98±0.47）%, which proved that quercetin inhibited increment of volume and weight of transplanted tumor in nude mice bodies.

Angiostatin inhibits pancreatic cancer cell proliferation and growth in nude mice

AIM: To observe the biologic behavior of pancreatic cancer cells in vitro and in vivo, and to explore the potential value of angiostatin gene therapy for pancreatic cancer.METHODS: The recombinant vector pcDNA3.1(+)-angiostatin was transfected into human pancreatic cancer cells PC-3 with Lipofectamine 2000, and paralleled with the vector and mock control. Angiostatin transcription and protein expression were determined by immunofluorescence and Western blot. The stable cell line was selected by G418. The supernatant was collected to treat endothelial cells. Cell proliferation and growth in vitro were observed under microscope. Cell growth curves were plotted.The troms-fected or untroms-fected cells overexpressing angiostatin vector were implanted subcutaneously into nude mice. The size of tumors was measured, and microvessel density count (MVD) in tumor tissues was assessed by immunohistochemistry with primary anti-CD34antibody.RESULTS: After transfected into PC-3 with Lipofectamine 2000 and selected by G418, macroscopic resistant cell clones were formed in the experimental group transfected with pcDNA 3.1(+)-angiostatin and vector control. But untreated cells died in the mock control. Angiostatin protein expression was detected in the experimental group by immunofluorescence and Western-blot. Cell proliferation and growth in vitro in the three groups were observed respectively under microscope. After treatment with supernatant, significant differences were observed in endothelial cell (ECV-304) growth in vitro. The cell proliferation and growth were inhibited. In nude mice model, markedly inhibited tumorigenesis and slowed tumor expansion were observed in the experimental group as compared to controls, which was parallel to the decreased microvessel density in and around tumor tissue.CONCLUSION: Angiostatin does not directly inhibit human pancreatic cancer cell proliferation and growth in vitro,but it inhibits endothelial cell growthin vitro. It exerts the anti-tumor functions through antiangiogenesis in a paracrine way in vivo.

Mast cell deficiency exacerbates inflammatory bowel symptoms in interleukin-10-deficient mice

AIM: To test the role of mast cells in gut inflammation and colitis using interleukin(IL)-10-deficient mice as an experimental model.METHODS: Mast cell-deficient(KitW-sh/W-sh) mice were crossbred with IL-10-deficient mice to obtain double knockout(DKO) mice. The growth, mucosal damage and colitis status of DKO mice were compared with their IL-10-deficient littermates.RESULTS: DKO mice exhibited exacerbated colitis compared with their IL-10-deficient littermates, as shown by increased pathological score, higher myeloperoxidase content, enhanced Th1 type pro-inflammatory cytokines and inflammatory signaling, elevated oxidative stress, as well as pronounced goblet cell loss. In addition, deficiency in mast cells resulted in enhanced mucosal damage, increased gut permeability, and impaired epithelial tight junctions. Mast cell deficiency was also linked to systemic inflammation, as demonstrated by higher serum levels of tumor necrosis factor α and interferon γ in DKO mice than that in IL-10-deficient mice.CONCLUSION: Mast cell deficiency in IL-10-deficient mice resulted in systematic and gut inflammation, impaired gut barrier function, and severer Th1-mediated colitis when compared to mice with only IL-10-deficiency. Inflammation and impaired gut epithelial barrier function likely form a vicious cycle to worsen colitis in the DKO mice.

Differentiation of Embryonic Stem Cells into Neurons and Retina-like Structure in Nude Mice

Purpose:To investigate the intraocular growth and biological characteristics of mice embryonic stem cells in nude mice.Methods:Murine embryonic stem cells(D3 cell line)were cultured and maintained in an undifferentiated state in vitro,then transplanted into the anterior chamber of nude mice.Mophological and immunohistochemical examinations were implemented.Results:Two to three days after transplantation,yellow-white floating granules,sheets and masses were seen inside the anterior chamber and vitreous cavity,and enlarged gradually,14-20days later,the mice were executed.Morphological examination showed that there were undifferentiated cells and some round or polygonal differentiated cells in anterior chamber and vitreous cavity.The morphology of these differentiated cells were similar to that of the retina.The cells were highly positive in NSE staining.Conclusion:The tranplanted embryonic stem cells cold grow in the eyes of nude mice with tendency to differentiate into neurons and retina-like structure.

Estrogen deficiency leads to telomerase inhibition, telomere shortening and reduced cell proliferation in the adrenal gland of mice

雌激素缺乏调停变老，但是内在的机制尚待充分坚定。我们这里报导 aromatase 的指向的混乱在老鼠引起的雌激素缺乏在 vivo 在肾上腺导致 telomerase 活动的重要抑制。基因表示分析证明当雌激素不在时， telomerase 颠倒 transcriptase (TERT ) 基因表示在肾上腺外皮和肾的萎缩与损害房间增长联合被减少。在 c 工具包积极的干细胞被识别在肾的外皮的实质占据。telomeres 的分析表明那雌激素缺乏比那在肾的外皮导致显著地更短的 telomeres 在野类型(WT ) 控制鼠标。为了推进，建立雌激素的原因的效果，我们在这些雌激素缺乏的动物进行了雌激素代替治疗。为 3 个星期的雌激素的管理在雌激素缺乏的老鼠恢复 TERT 基因表示， telomerase 活动和房间增长。因此，我们第一次，那雌激素缺乏在 vivo 在鼠标的肾上腺引起 TERT 基因表示， telomerase 活动， telomere 维护，和房间增长的抑制的数据表演，建议那 telomerase 抑制并且 telomere 弄短可以调停在肾上腺的房间增长拘捕，因此贡献雌激素在生理的条件下面的导致缺乏的老化。

Functional Mechanism of Resveratrol in Inhabiting Growth of Cells ls174t and Its Mechanism in Subcutaneously Transplanted Tumor of Nude Mice

To explore the functional mechanism of Resveratrol against colon cancer cells ls174t and the growth of colon cancer tissue of tumor-bearing mice, MTT method was used to observe the functions of resveratrol for inhibition against cells ls174t in vitro. Transmission electron microscope was used to observe the cell apoptosis. FCM assay was performed to measure the change of the cell apoptosis rate and of cell cycle. RT-PCR method was used to detect the expressions of bcl-2 and bax mRNA. Western blot method was used to detect the expressions of bcl-2 and bax protein. Ceils ls174t were transplanted subcutaneously to nude mice to observe the effect of resveratrol on the growth of subcutaneously transplanted tumor, RT-PCR method was used to detect the expressions of bcl-2 and bax mRNA in the tumor tissue. Western blot method was used to detect the expressions of bcl-2 and bax protein in the tumor tissue. Resveratrol has an effect of inhibiting proliferation of cells ls174t in vitro（P〈0.01）. It is able to induce the apoptosis of cells ls174t, causing the decrease in the expression of bcl-2 and the increase in the expression of bax. Resveratrol could inhibit the growth of subcutaneously transplanted tumor of nude mice（P〈0.05）, causing the decrease in the expression of bcl-2 and the increase in the expression of bax. Resveratrol can inhibit the growth of cells 174t and the growth of subcutaneously transplanted tumor. The mechanism is possibly related to the induction of the cell apoptosis and the regulation of bcl-2/bax expression.

Immuno-protective effect of tumor cell vaccine on Kunming mice bearing Ehrlich ascites tumor

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The recruitment of exogenous endothelial progenitor cells in lung tumor model of nude mice

Background and Objective: Endothelial progenitor cells (EPCs) play an important role in hypoxia-triggered tumor vasculogenesis. However, the homing of exogenous EPCs in tumors is still unclear. In this study, we investigated the recruitment of exogenous EPCs in human lung adenocarcinoma model of nude mice. Methods: EPCs labeled with green fluorescence protein (GFP) were transplanted into nude mice bearing human lung adenocarcinoma. The growth of tumor was observed. After the mice were killed, GFP-EPCs in different tissues were examined by fluorescence. The tumor tissues were stained for CD133, hypoxia-inducible factor-1alpha (HIF-1α), stromal cell-derived factor-1α (SDF-1α), and vascular endothelial growth factor receptor (KDR). Real-time polymerase chain reaction of CD133, HIF-1α, SDF-1α, and VEGF-1 were also performed. Results: The growth of tumor in EPC group was significantly faster than that in saline solution group (P < 0.05). Under fluorescence microscope, GFP-EPCs were strongly expressed in both tumor and bone marrow. EPCs were recruited to the tumor periphery to participate in tumor vasculogenesis. The expression of CD133, HIF-1α, and SDF-1 mRNA in tumor and bone marrow were significantly higher than that in the liver, spleen, and skin (P < 0.05). Conclusions: Exogenous EPCs can be recruited to tumor and accelerate tumor growth. Except tumor, bone marrow can also recruit EPCs.

Bioassay of Eucalyptus extracts for anticancer activity against Ehrlich ascites carcinoma(eac) cells in Swiss albino mice

Objective:To evaluate the antineoplastic activity of Eucalyptus extract(EUE) against Ehrlich ascites carcinoma(EAC)in Swiss albino mice.Methods:Preliminary examination of four plant extracts(namely Eucalyptus,Costus,Azadirachla.Feroniai has been done by observing the reduction ability of number of EAC cells in previously inoculated Swiss alliino mice.Among them as EuE showed maximum capability,the whole study has been conducted with EuE only. Important parameters viz.enhancement of life span,reduction of average tumor weight etc.have been studied.In addition the effects of EuE on hematological parameters in both normal and EAC inoculated mice have been measured.Effect of EuE on normal peritoneal cells has also been studied.Results:EuE reduced tumor burden remarkably.It reduced the tumor growth rate and enhanced the life span of EAC bearing mice noticeably.It reversed back the hematological parameters towards normal,reduced the Irasplanlability of EAC cells and enhanced the immunomodulatory effects in mice.The host toxic effect of EuE in mice is minimum and mostly reversible with time.All such data have been compared with those obtained by running parallel experiments with bleomycin at dose 0.3 mg/kg(i.p.).Conclusions:The Eucalyptus extract may be considered as a potent anticancer agent for advanced researches.

COMPARATIVE STUDY OF PHOSPHOTYROSYL PROTEIN PHOSPHATASE OF MICE NORMAL LIVER, REGENERATING LIVER, AND MICE H22A HEPATOMA ASCITES CELL

In regenerating liver of mice, marked increase of the activity of phosphotyrosyl protein phosphatase (PTPP) in cytosol was observed. The PTPP activity varied with time and reached the highest level between 24 to 48 hours after partial hepatectomy. In H22a cells the PTPP activity found in every subcellular fraction was lower than that of the normal liver. The PTPP activity was mostly concentrated in lysosomes of normal liver, but mainly distributed in nucleus, cytosol and microsome of regenerating liver. In H22a cells PTPP activity seemed distribute evenly. Five similar major PTPP peaks (I-V) were obtained on DEAE cellulose chromatography of cytosols from all three of liver cells studied. However, two additional PTPP peaks, a and b, were also obtained from cytosol of liver.

Antitumor Activities and Apoptosis-regulated Mechanisms of Fermented Barley Extract in the Transplantation Tumor Model of Human HT-29 Cells in Nude Mice

Objective A subcutaneous transplantation tumor model of human HT-29 cells was established in nude mice to study the anticarcinogenic activities and apoptosis-regulatory mechanistic effect of aqueous extract of fermented barley with Lactobacillus plantarum dy-1 （LFBE）. Methods HT-29 cells were transplanted via subcutaneous injection of 1 × 107cells into the right flank of each nude mouse. Then, nude mice were treated for 30 days with LFBE （high-dose 2 g·kg-1·d-1; low-dose 1 g·kg-1·d-1） and for 7 days with 5-fluorouracil （5-FU, 25 g·kg-1·d-1） by gavage and intraperitoneal injection, respectively. Results Tumor volume and weight decreased significantly in both groups of nude mice treated with LFBE. In addition, the cell apoptosis rate of the LFBE group was significantly higher than that of the control group and 5-FU groups as measured by the TUNEL assay. Moreover, the real-time fluorescent quantitative PCR and Western blot methods further confirmed these apoptosis-enhancing and growth-inhibiting effects. The involvement of LFBE in inducing apoptosis was confirmed by the expression of Bax, Bcl-2, caspase-3, and cyclin D1. Conclusion The results showed that LFBE could induce subcutaneous transplantation tumor apoptosis in nude mice and could be used as a natural nutrient supplement or chemopreventive agent in the treatment of human colon cancer.

IMMUNORESPONSES OF HUMANIZED SCID MICE TO HUMAN LUNG CANCER CELLS

ＩＭＭＵＮＯＲＥＳＰＯＮＳＥＳＯＦＨＵＭＡＮＩＺＥＤＳＣＩＤＭＩＣＥＴＯＨＵＭＡＮＬＵＮＧＣＡＮＣＥＲＣＥＬＬＳＣｈｅｎＬｉｚｈｅｎ；（陈力真），ＷａｎｇＳｈｕｈｕｉ；（王树蕙），ＺｈａｎｇＹｕｎ；（张云）ａｎｄＷａｎｇＳｈｉｚｈｅｎ；（王世真）（Ｉ...

Efficient production of chimeric mice from embryonic stem cells injected into 4-to 8-cell and blastocyst embryos

Background： Production of chimeric mice is a useful tool for the elucidation of gene function. After successful isolation of embryonic stem （ES） cell lines, there are many methods for producing chimeras, including co-culture with the embryos, microinjection of the ES ceils into pre-implantation embryos, and use of tetraploid embryos to generate the full ES-derived transgenic mice. Here, we aimed to generate the transgenic ES cell line, compare the production efficiency of chimeric mice and its proportion to yield the male chimeric mice by microinjected ES cells into 4- to 8-cell and blastocysts embryos with the application of Piezo-Micromanipulator （PMM）, and trace the fate of the injected ES cells. Results： We successfully generated a transgenic ES cell line and proved that this cell line still maintained pluripotency. Although we achieved a satisfactory chimeric mice rate, there was no significant difference in the production of chimeric mice using the two different methods, but the proportion of the male chimeric mice in the 4- to 8-cell group was higher than in the blastocyst group. We also found that there was no tendency for ES cells to aggregate into the inner cell mass using in vitro culture of the chimeric embryos, indicating that they aggregated randomly. Conclusions： These results showed that the PMM method is a convenient way to generate chimeric mice and microinjection of ES cells into 4- to 8-cell embryos can increase the chance of yielding male chimeras compared to the blastocyst injection. These results provide useful data in transgenic research mediated by ES cells.