Effect of Nimesulide on proliferation and apoptosis of human hepatoma SMMC—7721 cells

AIM:Cyclooxygenase-2(COX-2)has been suggested to be associated with carcinogenesis.We sought to investigate the effect of the selective COX-2 inhibitor,Nimesulide on proliferation and apoptosis of SMMC-7721 human hepatoma cells.METHODS:This study was carried out on the culture of hepatic carcinoma SMMC-7721 cell line Various concentrations of Nimesulide(0,200μmol/L,300μmol/L,400μmol/L)were added and incubated.Cell proliferation was detected with MTT colorimetric assay,cell proliferation was detected with MTT colorimetric assay,cell apoptosis by electron microscopy,flow cytometry and TUNEL.RESULTS:Nimesulide could significantly inhibit SMMC-7721 cells proliferation dose-dependent and in a dependent manner compared with that of the controla group.The duration lowerst inhibition rate produced by Nimesulide in SMMC-7721 cells was 19.06%,the highest inhibition rate was 58.49%,After incubation with Nimesulide for 72h,the most highest apotosis rate and apoptosis index of SMMC-7721 cells comparing with those of the control were 21.20%±1.62%,vs2.24%±0.26%and 21.23±1.78vs2.01±0.23(P<0.05).CONCLUSION:The selective COX-2 inhibitor,Nimesulide can inhibit the proliferation of SMMC-7721 cells and increase apoptosis rate and apoptosis index of SMMC-7721 cells,The apoptosis rate and the apoptosis index are dose-dependent.Under electron microscope SMMC-7721 cells incubated with 300 μmol and 400μmol Nimesulide show apoptotic characteristics With the clarification of the mechanism of selective COX-2 inhibitors,Thtese COX-2 selective inhibitors can become the choice of prevention and treatment of cancers.     

Apoptosis of hepatoma cells SMMC—7721 induced by Ginkgo biloba seed polysaccharide

AIM：To study the apoptosis of hepatoma cells SMMC-7721induced by polysaccharide isolated from Ginkgo biloba seed.METHODS:Ginkgo biloba seed polysaccharide(GBSP)was isolated by ethanol fractionation of Ginkgo biloba seed and purified by Sephadex G-200 chromatography,The purity of GBSP was verified by reaction with iodine-potassium iodide and ninhydrin and confirmed by UV spectrophotometer,cellulose acetate membrane electrophoresis and Separose 4B gel filtration chromatography,The Scanning Electron Microscope(SEM)and Flow Cytometry(FCM)were used to examine the SMMC-7721 cells with and without GBSP treatment at 500mg/ml for 36h.RESULTS:GBSP product obtained was of high purity with the average molecular weight of 1.86×10^5.Quantitative analysis of SMMC-7721cells in vitro with FCM showed that the percentages of G2-Mcells without and with GBSP treatment were17.01±1.28%and 11.77±1.50%(P<0.05).the debris ratio of the cells were0.46±0.12%and 0.06±0.06%(P<0.01).and the apoptosis ration of cells was3.84±0.55%and9.13±1.48%(P<0.010respectively,Following GBSP treatment,microvilli of SMMC-7721cells appeared thinner and the number of spherical cells increased markedly,Most significantly,the apoptosis bodies were formed on and around the sppherical cells treated with GBSP.CONCLUSION:GBSP could potentially induce the apoptosis of SMMC-7721cells.     

Chaga mushroom （Inonotus obliquus） induces G0/G1 arrest and apoptosis in human hepatoma HepG2 cells

AIM： To investigate the anti-proliferative and apoptotic effects of Chaga mushroom （Inonotus obliquus） water extract on human hepatoma cell lines, HepG2 and Hep3B cells. METHODS： The cytotoxicity of Chaga extract was screened by 3-[4,5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide （MTT） assay. Morphological observation, flow cytometry analysis, Western blot were employed to elucidate the cytotoxic mechanism of Chaga extract. RESULTS： HepG2 cells were more sensitive to Chaga extract than Hep3B cells, as demonstrated by markedly reduced cell viability. Chaga extract inhibited the cell growth in a dose-dependent manner, which was accompanied with G0/G1-phase arrest and apoptotic cell death. In addition, G0/G1 arrest in the cell cycle was closely associated with down-regulation of p53, pRb, p27, cyclins D1, D2, E, cyclin-dependent kinase （Cdk） 2, Cdk4, and Cdk6 expression. CONCLUSION： Chaga mushroom may provide a new therapeutic option, as a potential anticancer agent, in the treatment of hepatoma.     

Protective role of metallothionein （Ⅰ／Ⅱ） against pathological damage and apoptosis induced by dimethylarsinic acid

AIM: To better clarify the main target organs of dimethylarsinic acid toxicity and the role of metallothionein (MTs) in modifying dimethylarsinic acid (DMAA) toxicity. METHODS: MT-Ⅰ/Ⅱ null (MT^-/-) mice and the corresponding wild-type mice (MT^+/+), six in each group, were exposed to DMAA (0-750 mg/kg body weight) by a single oral injection.Twenty four hours later, the lungs, livers and kidneys were collected and undergone pathological analysis, induction of apoptolic cells as determined by TUNEL and Mr concentration was detected by radio-immunoassay. RESULTS: Remarkable pathological lesions were observed at the doses ranging from 350 to 750 mg/kg body weight in the lungs, livers and kidneys and MT^+/+ mice exhibited a relatively slight destruction when compared with that in dose matched MT/ mice. The number of apoptotic cells was increased in a dose dependent manner in the lungs and livers in both types of mice. DMAA produced more necrotic cells rather than apoptotic cells at the highest dose of 750 mg/kg,however, no significant increase was observed in the kidney.Hepatic Mr level in Mr^+/+ mice was significantly increased by DMAA in a dose-dependent manner and there was no detectable amount of hepatic MT in untreated MT^-/- mice. CONCLUSION: DMAA treatment can lead to the induction of apoptosis and pathological damage in both types of mice.MT exhibits a protective effect against DMAA toxicity.     

Expression and identification of recombinant soluble single—chain variable fragment of monoclonal antibody MC3

AIM: To generate soluble single chain variable fragments (ScFv) of monoclonal antibody MC3 recognizing colorectal and gastric carcinomas. METHODS: mRNA was isolated from the hybridoma cell line producing MC3 and the DNAs encoding variable domains of heavy and light chains (VH and VL) of the antibody were amplified separately by RT-PCR and assembled into ScFv DNA with a linker DNA. The ScFv DNA was ligated into the phagemid vector pCANTAB5E and the ligated sample was transformed into E.coli TG1.The transformed cells were infected with M13KO7 helper phage to yield recombinant phages. After two rounds of panning with gastric carcinoma cell line AGS highly expressing MC3-binding antigen, the phage clones displaying ScFv fragments of the antibody were selected by ELISA. 4 phage clones showing strong signal in ELISA were used to infect E.coli HB2151 to express soluble ScFvs. The soluble ScFvs were identified by Dot blot and Western blot, and their antigen-binding activity was assayed by ELISA. The VH and VL DNAs of the ScFv DNA derived from phage clone 19 were sequenced. RESULTS: The VH,VL and ScFv DNAs were about 340 bp, 320 bp and 750 bp respectively. After two rounds of panning to the recombinant phages, 18 antigen-positive phage clones were selected from 30 preselected phage clones by ELISA. All the soluble ScFvs derived from the 4 out of the 18 antigen-positive phage clones were about M(r)32000 and concentrated in periplasmatic space under the given culture condition. The soluble ScFvs could bind the antigen, and they shared the same binding site with MC3. The sequences of the VH and VL DNAs of the MC3 ScFv showed that the variable antibody genes belonged to the IgG1 subgroup,kappa-type. CONCLUSION: The soluble ScFv of MC3 is successfully produced, which not only provides a possible novel targeting vehicle for in vivo and in vitro study on associated cancers, but also offers the antibody a stable genetic source.     

Rosiglitazone enhances fluorouracil-induced apoptosis of HT-29 cells by activating peroxisome proliferator-activated receptor γ

AIM:To examine whether and how rosiglitazone enhances apoptosis induced by fluorouracil in human colon cancer(HT-29)cells.METHODS:Human colon cancer HT-29 cells were cultured in vitro and treated with fluorouracil and/or rosiglitazone.Proliferation and growth of HT-29 cells were evaluated by MTT assay and trypan blue exclusion methods,respectively.The apoptosis of HT-29 cells was determined by acridine orange/ethidium bromide staining and flow cytometry using PI fluorescence staining.The expressions of peroxisome proliferator-activated receptor γ(PPARγ),Bcl-2 and Bax in HT-29 cells were analyzed by Western blot.RESULTS:Although rosiglitazone at the concentration below 30 μmol/L for 72 h exerted almost no inhibitory effect on proliferation and growth of HT-29 cells,it could significantly enhance fluorouracil-induced HT-29 cell proliferation and growth inhibition.Furthermore,10 μmol/L rosilitazone did not induce apoptosis of HT-29 cells but dramatically enhanced fluorouracil-induced apoptosis of HT-29 cells.However,rosiglitazone did not improve apoptosis induced by fluorouracil in HT-29 cells pretreated with GW9662,a PPARγ antagonist.Meanwhile,the expression of Bax and PPARγ was up-regulated,while the expression of Bcl-2 was down regulated in HT-29 cells treated with rosiglitazone in a time-dependent manner.However,the effect of rosiglitazone on Bcl-2 and Bax was blocked or diminished in the presence of GW9662.CONCLUSION:Rosiglitazone enhances fluorouracil-induced apoptosis of HT-29 cells by activating PPARγ.     

Induction of apoptosis by TPA and VP—16 is through translocation of TR3

AIM：To investigate the role of TR3 in induction of apoptosis in gastric cancer cells.METHODS:Human gastric cancer cell line,MGC80-3,was used .Expression of TR3 mRNA and its protein was detected by Northern blot and Western blot Localization of TR3 protein was showed by imunofluorescence analysis under laser-scanning confocal microscope.Apoptotic morthology was observed by DAPL fluorescence staining,and apoptotic index was couted manong 10000 cells randomly Stable transfection assay was carried out by Lipofectamine.RESULTS:Treatment of MGC80-3 cells with TPA and VP-16 resulted in apoptosis,accompanied by the repression of Bcl-2 protein in atime-dependent manner,At the same time,TPA and VP-16 also up-regulated expression level of TR3 mRNA in MGC80-3 cells that expressed TR3 mRNA When antisense-TR3 expression vector was transfected into the cells,expression of TR3 protein was repressed.In this case,TPA and VP-16 did not induce apoptosis.In addition,TPA and VP-16-induced apotosis involved in translocation of TR3,IN MGC80-3 cells,TR3 localized concentrative in nucleus,after treatment of cells with TPA and Vp-16,TR3 translocated from nuclesu to cytosol obviously,However,When this nuclear translocaion was blocked by LMB apoptosis was not occurred in MGC80-3 cells even in the presence of TPA and VP-16.CONCLUSION:Induction of apoptosis by TPA and VP-16 is through induction of TR3 expression and translocation of TR3 from nucleus to cytosol,Which may be a novel signal pathway for TR3,and represent the new biological function of TR3 to exert its effect on apoptosis in gastric cancer cells.     

Effect of pseudolaric acid B on gastric cancer cells： Inhibition of proliferation and induction of apoptosis

AIM: To examine the effect of pseudolaric acid B on the growth of human gastric cancer cell line, AGS, and its possible mechanism of action. METHODS: Growth inhibition by pseudolaric acid B was analyzed using MTT assay. Apoptotic cells were detected using Hoechst 33258 staining, and confirmed by DNA fragmentation analysis. Western blot was used to detect the expression of apoptosis-regulated gene Bcl-2, caspase 3, and cleavage of poly (ADP-ribose) polymerase-1 (PARP-1). RESULTS: Pseudolaric acid B inhibited the growth of AGS cells in a time- and dose-dependent manner by arresting the cells at G2/M phase, which was accompanied with a decrease in the levels of cdc2. AGS cells treated with pseudolaric acid B showed typical characteristics of apoptosis including chromatin condensation and DNA fragmentation. Moreover, treatment of AGS cells with pseudolaric acid B was also associated with decreased levels of the anti-apoptotic protein Bcl-2, activation of caspase-3, and proteolytic cleavage of PARP-1. CONCLUSION: Pseudolaric acid B can dramatically suppress the AGS cell growth by inducing apoptosis after G2/M phase arrest. These findings are consistent with the possibility that G2/M phase arrest is mediated by the down-regulation of cdc2 levels. The data also suggest that pseudolaric acid B can trigger apoptosis by decreasing Bcl-2 levels and activating caspase-3 protease.     

抗细粒棘球绦虫成虫单克隆抗体的制备及鉴定

目的 建立能分泌与细粒棘球绦虫(简称包虫)成虫特异结合的抗包虫成虫单克隆抗体的杂交瘤细胞株。方法 用包虫成虫抗原免疫BALB／c小鼠后，获取免疫的脾细胞，与小鼠骨髓瘤细胞SP2／0融合。结果 ELISA方法筛选出1株能持续稳定分泌抗包虫成虫单克隆抗体的杂交瘤细胞株，1F5E3C9E9D5杂交瘤细胞株分泌的单克隆抗体可以与包虫成虫、棘球蚴结合，与囊液抗原呈边缘性阳性反应，而与泡球蚴EM2抗原呈阴性反应。冻存1个月后复苏，仍能稳定分泌。经免疫组织化学检测发现，该单克隆抗体与棘球蚴的生发层，棘球蚴原头节虫体呈阳性反应。结论 1F5E3C9E9D5是一株稳定的杂交瘤细胞株，所分泌的抗细粒棘球绦虫成虫单克隆抗体在粪抗原的检测方面有应用前景。     

Immunotherapy of hepatoma with a monoclonal antibody against murine endoglin

AIM: To explore the capability of a monoclonal antibody (mAb) against murine endoglin to inhibit tumor angiogenesis and suppression of hepatoma growth in murine models. METHODS: A monoclonal antibody against murine endoglin was purified by affinity chromatography and passively transfused through tail veins in two murine hepatoma models. Tumor volume and survival time were observed at three-day intervals for 48 d. Microvessels in tumor tissues were detected by immunohistochemistry against CD31, and angiogenesis in vivo was determined by alginate encapsulated assay. In addition, tumor cell apoptosis was detected by TUNEL assay. RESULTS: Passive immunotherapy with anti-endoglin mAb could effectively suppress tumor growth, and prolonged the survival time of hepatoma-bearing mice. Angiogenesis was apparently inhibited within the tumor tissues, and the vascularization of alginate beads was also reduced in the mice passively transfused with anti- endoglin mAb. In addition, increased apoptotic cells were observed within the tumor tissues from the mice passively transfused with anti-endoglin mAb. CONCLUSION: Passive immunotherapy with anti- endoglin mAb effectively inhibits tumor growth via inhibiting tumor angiogenesis and increasing tumor cell apoptosis, which may be highly correlated with the blockage of endoglin-related signal pathway induced bya nti-endoglin mAb.     

氨基端前B型钠尿肽单克隆抗体的制备及其鉴定

目的通过经典单克隆抗体制备技术制备氨基端前B型钠尿肽(NT-pro BNP)单克隆抗体,将单克隆抗体进行特异性和亲和力鉴定,并用于免疫学检测试剂盒的开发。方法用带His-Trx-标签蛋白的NT-pro BNP免疫Balb/C小鼠制备单克隆抗体。使用PEG1500化学融合法将小鼠脾细胞与骨髓瘤细胞SP2/0进行融合。用不带标签的NT-pro BNP对获取的杂交瘤细胞进行反复多次克隆筛选,将稳定分泌抗体的杂交瘤细胞株经腹腔注射Balb/C小鼠诱导产生腹水,经蛋白G亲和纯化后获得NT-pro BNP单克隆抗体并对其进行亲和力和特异性进行鉴定。结果成功进行细胞融合并获得4株稳定分泌单克隆抗体的杂交瘤细胞株,抗体分泌亚型为Ig G1,可特异性识别NT-pro BNP。经腹腔体内诱生法和蛋白G亲和纯化,获得高质量NT-pro BNP单克隆抗体,经ELISA检测抗体效价在1∶10~5以上。结论通过传统经典单克隆抗体制备技术成功制备NT-pro BNP单克隆抗体,为NT-pro BNP检测试剂盒的研发奠定了基础。     

Preparation of monoclonal antibody against Angiostrongylus cantonensis antigen

Monoclonal antibodies (MAb) against A cantonensis were produced through fusion of immunised spleen cells from BALB/c mice with NS-1 myeloma cells at a ratio of 10:1. The successful fusion rate on the 3rd day of fusion was 90.1%. Ten MAb were characterised, six of which were IgG1 and the remaining four were IgG2a, IgG2b, IgM and IgA respectively. Among 6 IgG1 MAb, four were A. cantonensis-specific, of which three reacted to adult worm antigen only and one reacted to both adult worm and juvenile worm antigens. Two other IgG1 MAb showed cross-reaction with other helminthic antigens of Toxocara canis. Ascaris suum. Paragonimus westermani, Dirofilaria immitis, Anisakis Spp, Gnatostoma Spinigerum and Clonorchis sinensis.     

抗弓形虫单克隆抗体的制备鉴定及应用

目的制备抗弓形虫全抗原、天然P30、重组P30的单克隆抗体（McAb），为抗原提纯、弓形虫病诊断及亚单位疫苗的研制奠定基础。方法用弓形虫全抗原、天然P30、重组P30分别免疫BALB／c小鼠，取脾细胞与骨髓瘤细胞融合，筛选稳定分泌高滴度McAb的杂交瘤细胞株。用ELISA法测定McAb亚类和效价；通过SDS—PAGE和Western blot进行特异性鉴定;Giemsa染色观察杂交瘤细胞的染色体数目；利用电镜及IFAT观察McAb对弓形虫的杀伤作用及其作用位点。结果筛选出3株（4810、283、1H6）特异性较好的杂交瘤细胞株。Western blot结果显示，283、1H6产生的MeAb与弓形虫全抗原的阳性反应带均在30kDa处，4810反应带主要在22kDa处；283，1H6，4810MeAb的效价（ELISA）分别为：1：102400、1：51200、1：12800；283、1H6为IgM，4810为IgG2b；3株细胞的染色体数均在100条以上。电镜观察到McAb作用后的弓形虫虫体聚集、肿胀，表面出现缺口和空洞，虫体变形破碎、膜变厚。抗弓形虫全抗原及天然P30的McAb能准确识别重组体pET-30a—ROP2、pET-30a-P30的表达蛋白。结论抗弓形虫全抗原、P30抗原的McAb均对弓形虫具有明显的杀伤作用，能准确识别P30抗原，可应用于弓形虫病诊断、抗原鉴定及亚单位疫苗的研制。     

Blocking of pregnancy in mice by immunization with anti-idiotype directed against monoclonal anti-progesterone antibody.

Passive transfer of monoclonal anti-progesterone antibodies shortly after mating blocks the onset of pregnancy in different species (mouse, rat, and ferret). Here we report that BALB/c mice can be actively immunized against progesterone, and hence against pregnancy, by means of rabbit anti-idiotypic antibodies specific for a mouse monoclonal anti-progesterone antibody, DB3. Some of the anti-idiotypic antibodies reacted with the steroid-combining site on the DB3 molecule. In response to repeated anti-idiotypic immunization, mice produced serum anti-progesterone antibodies (up to 100 micrograms/ml) that resembled DB3 in idiotypy, affinity, and specificity for progesterone and other steroid ligands. Thus an anti-idiotype can mimic the antigenicity of a steroid hormone with a high degree of accuracy. Compared with immunization with a progesterone-bovine serum albumin conjugate, the anti-progesterone response to anti-idiotype was considerably lower and clonally restricted. When mated after completion of the immunization course, the fertility rate of anti-idiotype-immunized mice was reduced to 30% from a control level of 91%. The anti-fertility effect was correlated with the circulating anti-progesterone concentration in individual animals and persisted for 4 or 5 estrous cycles. Active immunization with progesterone-bovine serum albumin was a highly effective means of rendering mice infertile; it reduced the fertility rate to zero over 16 or 17 estrous cycles. Our results suggest that anti-idiotypes may form the basis of contraceptive vaccines.     

Production of a monoclonal antibody against the Snow Mountain agent of gastroenteritis by in vitro immunization of murine spleen cells.

The Snow Mountain agent (SMA) is a 27- to 32-nm noncultivatable virus that causes acute gastroenteritis in humans. SMA is morphologically similar to but immunologically distinct from the Norwalk agent. SMA has been partially purified from the stool of experimentally infected volunteers and contains a single structural protein of Mr 62,000 as well as one or more non-virion-associated soluble proteins. Further characterization of this important human pathogen and other Norwalk-like viruses has been hindered by the lack of reagents with which to study them. To further characterize SMA, we developed a monoclonal antibody to SMA using in vitro immunization--a technique that permitted use of small quantities of antigen for immunization. The monoclonal antibody, SM-4, was specific for SMA and did not react with the Norwalk or Hawaii agents. In addition, SM-4 reacted with purified virion but not with the soluble protein. SM-4 also blocked the ability of labeled postinfection human IgG to bind to purified virion. Finally, both SM-4 and human postinfection sera specifically recognized the Mr 62,000 virion-associated protein. Thus, SM-4 is directed against an epitope present on the SMA structural protein that is not shared by the Norwalk or Hawaii agents and that is not present on the soluble protein. The availability of a monoclonal antibody against SMA should facilitate further purification and characterization of this agent. The techniques utilized in these studies provide a method for the production of additional monoclonal antibodies to this group of viruses and also should be useful for the study of other occult viral agents.     

抗人DR5单克隆抗体诱导肝癌细胞株SMMC-7721凋亡实验研究

目的探讨抗人DR5单克隆抗体对肝癌细胞株SMMC-7721致凋亡作用。方法常规培养肝癌细胞SMMC-7721,通过MTT法检测细胞抑制作用,流式细胞术定量分析凋亡细胞率。结果抗人DR5单抗能够诱导肝癌SMMC-7721细胞凋亡,在抗人DR5单抗浓度2μg/ml作用48h,对肝癌SMMC-7721细胞的杀伤率可达50.10%,增加抗人DR5单抗浓度细胞凋亡作用无明显增加。结论抗人DR5单抗能够诱导肝癌细胞SMMC-7721凋亡,以死亡受体为靶点的抗体制剂为肝癌治疗提供新途径。     

抗扎伊尔型埃博拉病毒单克隆抗体的制备和鉴定

目的 采用扎伊尔型埃博拉病毒核蛋白(EBOV NP)的合成多肽为免疫原制备鼠源单克隆抗体,并用4种埃博拉流行株核蛋白基因的真核表达蛋白对制备的单克隆抗体进行特异性分析。方法 用人工合成的扎伊尔型埃博拉病毒核蛋白多肽免疫动物,进行细胞融合后筛选阳性杂交瘤细胞。构建埃博拉病毒4种亚型的真核表达载体转染HEK293T细胞以表达目的蛋白,再以真核表达蛋白为抗原,用免疫印迹方法分析扎伊尔型埃博拉病毒单克隆抗体的特异性。结果 完成了抗扎伊尔型单克隆抗体的制备,共得到能分泌抗扎伊尔型埃博拉病毒单克隆抗体的杂交瘤细胞12株,小鼠腹水抗体效价介于1∶10⁴~1∶10⁵,其中3株杂交瘤细胞株分泌的抗扎伊尔型埃博拉病毒单克隆抗体与其他3种亚型无交叉反应,抗体效价高、特异性好。结论 成功制备抗扎伊尔型埃博拉病毒单克隆抗体,为建立快速检测埃博拉病毒的方法奠定基础。     

肝癌特异性谷氨酰转肽酶单克隆抗体的制备与初步应用

目的 制备抗肝癌特异性谷氨酰转肽酶(GGT)单克隆抗体及研究此单克隆抗体的临床应用。 方法 以纯品GGT-Ⅱ免疫BALB/C小鼠,取免疫鼠脾细胞与SP2/0骨髓瘤细胞融合以制备能产生抗GGT-Ⅱ单克隆抗体的杂交瘤细胞株;以此抗GGT-Ⅱ单克隆抗体采用竞争抑制性ELISA检测人血清GGT-Ⅱ。 结果对融合细胞阳性孔反复克隆后获得1株稳定分泌抗GGT-Ⅱ单克隆抗体的小鼠杂交瘤细胞系:2G4F6B2,此单克降抗体为IgG1类,与GGT-I无交叉反应。以此单克隆抗体测定人血清GGT-II的结果与聚丙酰胺梯度电泳法相符。 结论 研究制备的抗GGT-Ⅱ单克隆抗体特异性高,可应用于临床检测人血清中GGT-Ⅱ。