This study was purposed to investigate the effect of different heat stress conditions on expression level of heat shock protein gp96 in K562 cell line of chronic myeloid leukemia in order to provide experiment basis for seeking optimal heat stress condition increasing extraction amount of gp96 from K562 cells. The expression changes of gp96 in K562 cell line was detected by immunocytochemistry under 38, 40, 42, 44, 46 and 48 degrees C for 30 minutes in water, by flow cytometry under 40, 44, 48 and 52 degrees C for 30 minutes in water, by Western blot under 40, 44, 48 and 52 degrees C for 30 minutes in water. Immunocytochemistry assay showed that gp96 existed mainly in cytoplasm. The peak of gp96 expression was at 30 minutes after 48 degrees C in water. The result of flow cytometry was consistent to immunocytochemistry detection results under temperatures 40, 44, 48 and 52 degrees C (P < 0.01). Western blot showed that detection result was the same as the immunocytochemistry and flow cytometry detections. In conclusion, the expression of gp96 in K562 cell line reached peak at 30 minutes after 48 degrees C in water. This condition may be an effective preparative condition for extraction of gp96 from K562 cells.

目的:构建人骨形态发生蛋白2反转录病毒表达载体,探讨以转人骨形态发生蛋白2基因骨髓基质细胞为种子细胞的组织工程骨修复骨缺损的可行性.方法:实验于2001-05/2003-01在北京大学医学部完成.①采用DNA重组技术,将骨形态发生蛋白2cDNA克隆到pLXSN反转录病毒质粒,经酶切及测序鉴定正确后,包装成为pLXSN/BMP2重组反转录病毒.取15 d龄BALB/c系小鼠5只用于骨髓基质细胞的提取.将重组病毒感染小鼠骨髓基质细胞,经筛选后,聚合酶链反应鉴定人骨形态发生蛋白2cDNA在转基因骨髓基质细胞中的整合情况.②转基因骨髓基质细胞的骨形态发生蛋白2表达情况采用免疫印渍杂交和核糖核酸印渍杂交技术检测.转基因骨髓基质细胞碱性磷酸酶的表达采用钙-钴法染色检测,用以评定表达骨形态发生蛋白2的生物活性.③取6周龄BALB/c小鼠6只,将转基因骨髓基质细胞与羟基磷灰石构建人工骨植于小鼠骨骼肌内,体内成骨情况予组织学观察评估.结果:①pLXSN/BMP2质粒的鉴定结果:经酶切及DNA测序证实重组pLXSN/BMP2序列正确.②重组病毒的滴度测定结果:包装的病毒滴度为(6～8)×105cfu/mL.③基因整合及表达的分析:聚合酶链反应证实转基因骨髓基质细胞基因组中有骨形态发生蛋白2 cDNA整合;核糖核酸印渍杂交和蛋白免疫印渍杂交证实转基因骨髓基质细胞稳定表达人骨形态发生蛋白2;钙-钴法染色证实转基因骨髓基质细胞碱性磷酸酶阳性.④小鼠体内成骨情况:甲苯胺蓝染色的组织切片镜下观察显示转骨形态发生蛋白2基因骨髓基质细胞构成人工骨可见少量软骨细胞及软骨基质存在,软骨细胞呈巢状分布.结论:成功地构建了pLXSN-BMP2重组反转录病毒载体,该载体不仅有效地表达骨形态发生蛋白2蛋白,表达的骨形态发生蛋白2能够诱导未分化的骨髓基质细胞向成骨细胞方向分化,且在体内肌肉环境下能促进成骨.

Objective To establish an immortalized marrow stromal cell line for basic and clinical research of neuroscience use. Methods Human telomerase reverse transcriptase (hTERT) cDNA was transferred into human marrow stromal cells (hMSCs) by retroviral vector pLPChTERT. PCR, RT-PCR, and immunocytochemical methods were used to detect integration and expression of ectopic hTERT gene. Subcultured hMSC-hTERT cells were induced to differentiate into neuron-like cells usingβ-mercaptoethanol, trans-retinoic acid, forskolin, and basic fibroblast growth factor (bFGF). Results Expression of hTERT gene can be detected on DNA, RNA, and at protein level. hMSC-hTERT cells expressed neuron-specific makers NeuN and tubulin β-Ⅲ after induction. The 82nd passage of hMSC-hTERT cells has been obtained so far. Conclusion Ectopic hTERT gene has been stably integrated and expressed in hMSCs, therefore, immortalized hMSC-hTERT cell line is successfully established. Under certain conditions, hMSC-hTERT cells can be differentiated into neuron-like cells.

Objective To investigate the expression of HSP 70 mRNA in transitional cell carcinoma of bladder and association of HSP 70 mRNA with cell apoptosis. Methods In situ hybridization histochemistry(ISHH) was applied to detect HSP 70 mRNA expression in 60 patients with transitional cell carcinoma of bladder,and cell apoptosis was evaluated by means of terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end labeling technique(TUNEL). Results Of 60 cases of bladder transitional cell carcinoma,HSP 70 mRNA expression was 56.7%(34/60),and it increased with grade and stage progressing( P 0.05). There were significant differences( P 0.05) between G_1,G_2 and G_3 reciprocally,and the relationship of HSP 70 mRNA expression to stage had the same result with grade.Apoptotic indices(AIs) by TUNEL decreased significantly ( P 0.05) while malignancies increased.HSP 70 mRNA expression was negatively correlated with AIs(r_s=-0.685, P 0.05). Conclusions The HSP 70 mRNA expression increases but AIs decreases with malignancies progressing.It is implied that HSP 70 could inhibit cell apoptosis in the transitional cell carcinoma of bladder.

OBJECTIVE:To further study the effect of apoptin in inducing cancer cell specific apoptosis and the possible applications in cancer therapy.METHODS:Apoptin gene was amplified by PCR and inserted into pcDNA3.1(+) with a FLAG tag in front of the multi-cloning-site. Apoptin gene with the FLAG tag was sub-cloned into an adenovirus vector. Several cancer cell lines were transfected with pcDNA3.1/FLAG/apoptin or infected with apoptin containing recombinant adenoviruses to study the morphologic changes. Ad/apoptin infected cells were also analyzed by flowcytometry after staining with PI.RESULTS:Expressed apoptin was localized in the nucleus of cancer cells. Chromatin condensation occurred 2 or 3 days after Ad/apoptin(+) infection. Cell number in G(2)-M phase increased dramatically after Ad/apoptin(+) infection.CONCLUSION:Apoptin can induce cell cycle G(2)-M arrest and chromatin condensation in cancer cells.

热休克蛋白(HSP)是具有高度保守性的一族蛋白,广泛存在于原核和真核生物,gp96是HSP家族中的一员,近年在研究肿瘤免疫激活原理的过程中发现它参与机体特异的抗肿瘤免疫反应[1].Srivastava等[2]实验显示,从肿瘤组织中提取的gp96能激发出抗该肿瘤的排斥反应.

This study was to establish the method of purifying heat shock protein GP96 from K562 cells and explore the differentiation and function of human DC influenced by heat shock prolein (HSP). Using ammonium sulfate precipitation, conA-sepharose affinity chromatography and DEAE-sephacel anion exchange chromatography GP96 from K562 cells lysate was isolated and purified. The identification of the purified protein was controlled by Western blot with anti-human GP96 IgG. Human dendritic cell derived from peripheral blood mononuclear cell were cultured with purified GP96. The phenotype changes of DC were analyzed by flow cytometry and MLR was detected by MTT. The results showed that 60-80 microg GP96 was purified successfully from 1 x 10(10) K562 cells. DC stimulated with HSP-GP96 had higher expression rates of CD83, CD86, HLA-DR and lower expression rates of CD1a and had stronger ability to induce T cells proliferation. It is concluded that heat shock protein GP96 can be isolated and purified from K562 cells and could induce maturation of dendritic cell. HSP-DC vaccine show stronger ability to induce T cell proliferation than DC.

To explore the mechanism of transforming growth factor-beta1 (TGF-beta1) effect on umbilical cord blood mononuclear cells proliferation and apoptosis, 5-bromo-2'-deoxyurine (BrdU) incorporation assay was adopted to detect effect of TGF-beta1 on synthesis of DNA in cells. Western blot method was used to examine effect of TGF-beta1 on expression of cyclin A, Cyclin D1, CDK2 and CDK4 in G1 phase of cell cycle. Giemsa staining and flow cytometry (FCM) were performed to detected effect of TGF-beta1 on cell apoptosis. The results showed that (1) after culture of cells with IMDM containing 10% FBS, 10% FBS + 1 ng/ml TGF-beta1, 10% FBS + 2 ng/ml TGF-beta1 or 10% FBS + 5 ng/ml TGF-beta1 for 12 hours the OD values of TGF-beta1 group were significantly lower than control group (P <0.01); after culture for 24 hours the OD values of 1 ng/ml TGF-beta1 group had no significant difference compared with control group (P >0.05), but the OD values of 2 ng/ml and 5 ng/ml TGF-beta1 groups were significantly lower than control group (P <0.05). (2) 2 ng/ml TGF-beta1 could significantly inhibit the production of cyclin A, cyclin D1, CDK2 and CDK4, the protein levels were significantly lower than control group. (3) when the cells were co-cultured with 2 ng/ml TGF-beta1 for 12 and 24 hours, Giemsa staining and FCM detection could display typical apoptosis, the apoptosis rates were 14.42% and 31.98%, while apoptosis rate in control were 4.71% and 5.76%. It is concluded that TGF-beta1 can inhibit production of G1 cyclins and CDKs of umbilical cord blood mononuclear cells, arrest cells in the G1 phase of cell cycle and induce cell apoptosis. Thus, TGF-beta1 may be an important negative modulator in hematopoiesis.

OBJECTIVE:To investigate whether the heat shock protein (HSP) 70 antisense oligomers can enhance the sensitivity of bladder cancer cell EJ to mitomycin C.METHODS:The HSP70 mRNA of EJ cells was blocked by the 10 micromol/L HSP70 antisense oligomers, while its effect on cell growth was evaluated by methyl thiazolyl tetrazolium (MTT) and colony forming ability test.RESULTS:The HSP70 expressions in HSP70 antisense treated group were lower than the corresponding sense and nonsense treated groups (P < 0.01). While, the increased sensitivity of EJ to mitomycin C was found in antisense treated group, compared with the corresponding sense and nonsense treated groups (P < 0.01).CONCLUSION:The sensitivity of bladder cancer cell EJ to mitomycin C was enhanced by the blockage of the HSP70 expression.

腺病毒载体广泛应用于基因治疗和转基因研究,目前常用的E1/E3缺失型复制缺陷腺病毒载体虽然失去了病毒复制必需的E1基因,但载体上的其它病毒基因仍能在宿主细胞内表达.为研究这些基因对细胞的毒性作用,选择了3种携带没有明显细胞毒性外源基因的腺病毒载体,观察感染2种肿瘤细胞后细胞核形态改变,并用流式细胞仪检测细胞周期及凋亡情况.发现大剂量重组缺陷型腺病毒感染细胞后引起细胞变圆,核增大,细胞周期阻滞于G2/M期,继而染色质凝聚,细胞发生坏死或凋亡;各种腺病毒载体造成G2/M阻滞所需感染量不同,但都随时间延长和感染量增加而加重.这些结果提示腺病毒基因对细胞的影响是多方面的,在以此类病毒载体进行基因转移和基因治疗的研究中,精确滴定病毒滴度和转导效率非常重要,腺病毒基因表达造成的毒副作用给此类研究增加了变数.

利用基因转移技术,将mdr1基因转导造血干细胞,利用P170的泵出功能,可以保护干细胞免受细胞毒性药物的损伤.这种方案已得到美国FDA的认可进入临床实验阶段[1].我们将脐带血CD34+细胞作为基因转移的靶细胞,探讨体外基因转移的技术和程序.

Objective To observe the distribution of Alu I/D polymorphism of angiotensin converting enzyme(ACE) gene and A 1166-C single nucleotide polymorphism (SNP) of angiotensin Ⅱ type 1 receptor (AT 1R) gene in normal Chinese population. Methods The frequency of ACE genotype and A 1166-C SNP of AT 1R genotype were detected by using PCR-RFLP in normal Chinese people. Results In normal people of China ACE genotype the frequency of Ⅱ was 37。0%,ID was 40。5%,DD was 22。5%, The allele gene frequency of I was 57。3%,D was 42。7%;A 1166-C SNP of AT 1R genotype frequency of AA was 76。5%,AC was 23。4%, CC was 0。1%。The allele gene frequency of A was 88。2%, C was 11。8%。 Conclusion The frequency of ACE gene I/D Polymorphism and AT 1R gene A 1166-C SNP were varied significantly among different races and people。

基因转移技术的发展使外源耐药基因转染造血干细胞成为可能,为化疗药物带来的严重骨髓抑制提供了解决办法,并且作为选择性标志基因,在其它基因治疗中发挥重要作用.

树突状细胞(DC)是激活初始T细胞和维持细胞免疫反应最重要的抗原提呈细胞(APC),IFN-α在细胞及体液免疫中起着重要作用.然而目前对IFN-c在DC发育和功能方面作用的相关研究不多.我们在常规培养条件中加入不同剂量的IFN-α,以观察IFN-α对培养的DC表型和功能的影响,并通过MTT法检测成熟DC对同种异体T淋巴细胞增殖能力的作用、

OBJECTIVE:To explore the effect of p21(WAF1) on the proliferation and the sensitivity to Vp16 of leukemia cell line K562.METHODS:A p21(WAF1) retroviral expression vector pLXSN-p21(WAF1) was constructed by FuGENE 6, pLXSN-p21(WAF1) and pLXSN-neo, and transfected into p21(WAF1) defect leukemia cell line K562. After selected with G418, K562-p21(WAF1) cell clones that stably expressed p21(WAF1) were isolated. The ectopic expressions of p21(WAF1) mRNA and protein in K562-p21(WAF1) were identified by RT-PCR and Western b1ot. The cell growth rate was tested by trypan blue dye, the cell cycle by FCM and the sensitivity to Vpl6 by cell count and MTT assay.RESULTS:The expression of p21(WAF1) protein and mRNA could be detected in K562-p21(WAF1) cells. A strong inhibition of cell proliferation was observed in K562-p21(WAF1) cell as compared with that of the control. The cell number in G(0)/G(1) phase was remarkably increased. The sensitivity to Vpl6 decreased, the IC (50) of K562-neo cells was (56.4 +/- 6.5) microgram/ml, and that of K562-p21(WAF1) cells was (131.0 +/- 8.7) microgram/ml (P < 0.01).CONCLUSION:p21(WAF1) can inhibit the proliferation of leukemia cell and decrease its sensitivity to Vp16.

To produce HLA-B2704 transgenic mice by microinjection,286 Kunming female mice were induced to give superovulation by injecting hormone.The fertilized eggs were collected and microinjected with genomic DNA fragment containing HLA-B2704 DNA into their pronucleus.After microinjection,the survial and two-cell fertilized eggs were implanted into the oviducts of pseudopregnant female mice to let them develop and produce progeny.The intergration of the target gene in F0 and F1 mice was analyzed by PCR.The expression of HLA-B2704 transgene at mRNA level was comfirmed by RT-PCR.8 411 fertilized eggs were collected.6 609 of them were microinjected.The number of survial and two-cell eggs was 4 277 after microinjection.The survial rate was 64.7%.These eggs were implanted into 153 pseudopregnant female mice,26 of which were pregnant and 101 offsprings were obtained at last.PCR analysis showed that genomes of 10 F0 transgenic mice were integrated with the HLA-B2704 gene.The integration rate was 9.9%.Among 78 F1 of positive F0 transgenic mice,15 F1 mice carried the HLA-B2704 gene.HLA-B2704 gene was expressed at the mRNA level in skin,colon,testicle and spleen tissues of the transgenic mice.In the positive HLA-B2704 transgenic mice,six mice were detersile.One mouse showed tumescent at podosoma and toes.Two mice appeared photophobia.One mouse arised diarrhea.The transgenic mice carrying the HLA-B2704 genotype and phenotype were produced successfully.

为观察普伐他汀和辛伐他汀对载脂蛋白E缺陷小鼠主动脉粥样斑块形成的影响及主动脉壁血管细胞粘附分子-1表达的影响,将载脂蛋白E缺陷小鼠分为普伐他汀组(每天10 mg/kg)、辛伐他汀组(每天5 mg/kg)和阳性对照组(等量生理盐水),从主动脉血管根部连续切片,常规HE染色,计算机图像扫描,分析主动脉粥样硬化斑块的面积和斑块占管腔面积等;采用免疫组织化学及Western杂交方法测定主动脉壁血管细胞粘附分子-1表达.结果发现,除降胆固醇作用外,普伐他汀和辛伐他汀皆延缓斑块形成,与对照载脂蛋白E缺陷小鼠比,用药组小鼠的主动脉粥样斑块明显缩小;普伐他汀和辛伐他汀还明显抑制载脂蛋白E缺陷小鼠主动脉壁血管细胞粘附分子-1的表达;其中2药对14和24周龄小鼠主动脉壁血管细胞粘附分子-1表达的抑制作用强于34周龄小鼠.结果提示,普伐他汀和辛伐他汀可延缓或缩小载脂蛋白E缺陷小鼠主动脉粥样斑块的形成,抑制或下调主动脉壁血管细胞粘附分子-1表达,其效果与降胆固醇作用不成比例,可能是独立于调脂作用以外的抗动脉粥样硬化机制.

To establish a quantitative assay for telomerase activity and analyze the telomerase activity in peripheral blood mononuclear cells (PBMNC) from patients with acute leukemia, a fluorescent dye, PicoGreen, was added to the products after telomere repeat amplification protocol. The samples were excited at 480 nm and the fluorescence emission intensity was measured at 520 nm using a spectrofluorometer. Telomerase activity was detected in PBMNCs from 20 cases of normal individuals and 25 patients with acute leukemia. The results showed that the fluorescence of PicoGreen binding to double-stranded DNA specifically was enhanced with increase of DNA quantities. In conclusion, the met hod is rapid, simple and quantitative, the telomerase activities of PBMNCs from acute leukemia patients are significantly higher than that of the normal controls.

OBJECTIVE:To construct, package, and titrate vectors that express antitumor genes using adeno-associated virus (AAV) containing human alpha-fetoprotein (AFP) promoter.METHODS:Recombinant AAV vectors were constructed by blunted ligation of plasmids rAAV-AFP-GFP and pTR-UF5 and p16 cDNA and p21 cDNA. Packaging cell lines, 293 and C12 cells, were transfected with rAAV vectors, plasmid PspRC72, and adenovirus cosmid pAdc with the help of LipofectAMINE and then infected by wild adenovirus 48 hours later. The total viral particle titre was measured by PCR. Electron microscopy was used to observe the morphology of rAAV. C12 cells were infected with rAAV and wild adenovirus (MOI 10) and the titre of infectious virus was measured. by RCA method. Hepatocellular carcinoma cells were cultured and infected with rAAV containing green fluorescent protein (GFP), the infectious rate was observed 48 hours later.RESULTS:Recombinant vectors, rAAV-AFP-p16, rAAV-AFP-p21, rAAV-CMV-p16, and rAAV-CMV-p21, were constructed and verified by DNA sequencing and enzyme digestion. The total viral titre was 10(13) to 10(14)/ml. The titre of infectious rAAV containing GFP was 5 x 10(10)/ml. Electron microscopy showed that the rAVV was round and plaqued with a diameter between 20 and 30 nm. When the MOI was 100, the infectious rate of hepatocellular carcinoma cells was at least 70%. Four rAAV vectors carrying p16 or p21 genes have been constructed. rAAV packaging and titration methods were developed to produce highly-purified AAV stocks. These approaches help in research of gene therapy in liver cancer.