Suppression of P-gp induced multiple drug resistance in a drug resistant gastric cancer cell line by overexpression of Fas

AIM To observe the drug sensitizing effect andrelated mechanisms of fas gene transduction onhuman drug-resistant gastric cancer cellSGC7901/VCR(resistant to Vincristine).METHODS The cell cycle alteration wasobserved by FACS.The sensitivity of gastriccancer cells to apoptosis was determined by invitro apoptosis assay.The drug sensitization ofcells to several anti-tumor drugs was observedby MTT assay.Immunochemical method wasused to show expression of P-gp and Topo Ⅱ ingastric cancer cells.RESULTS Comparing to SGC7901 and pBK-SGC7901/VCR,fas-SGC7901/VCR showeddecreasing G2 cells and increasing S cells,theG2 phase fraction of pBK-SGC7901/VCR wasabout 3.0 times that of fas-SGC7901/VCR,but Sphase fraction of fas-SGC7901/VCR was about1.9 times that of pBK-SGC7901/VCR,indicatingS phase arrest of fas-SGC7901/VCR.FACS alsosuggested apoptosis of fas-SGC7901/VCR,fas-SGC7901/VCR was more sensitive to apoptosisinducing agent VM-26 than pBK-SGC7901/VCR.MTT assay showed increased sensitization offas-SGC7901/VCR to DDP,MMC and 5-FU,butsame sensitization to VCR according to pBK-SGC7901/VCR.SGC7901,pBK-SGC7901/ VCRand fas-SGC7901/VCR had positively stainedTopo Ⅱ equally.P-gp staining in pBK- SGC7901/VCR was stronger than in SG07901,but there was little staining of P-gp in fas.SGC7901/VCR.CONCLUSION fas gene transduction couldreverse the MDR of human drug-resistant gastriccancer cell SGC7901/VCR to a degree,possiblybecause of higher sensitization to apoptosis anddecreased expression of P-gp.

Down-regulation of Hsp90 could change cell cycle distribution and increase drug sensitivity of tumor cells

AIM:To construct Hsp90 antisense RNA eukaryotic expression vector, transfect it into SGC7901 and SGC7901/VCR of MDR-type human gastric cancer cell lines, HCC7402 of human hepatic cancer and Ec109 of human esophageal cancer cell lines, and to study the cell cycle distribution of the gene transected cells and their response to chemotherapeutic drugs.METHODS:A 1.03kb cDNA sequence of Hsp90beta was obtained from the primary plasmid phHSP90 by EcoR I and BamH I nuclease digestion and was cloned to the EcoR I and BamH I site of the pcDNA by T4DNA ligase and an antisense orientation of Hsp90beta expression vector was constructed. The constructs were transfected with lipofectamine and positive clones were selected with G418. The expression of RNA was determined with dot blotting and RNase protection assay, and the expression of Hsp90 protein determined with western blot. Cell cycle distribution of the transfectants was analyzed with flow cytometry, and the drug sensitivity of the transfectants to Adriamycin (ADR), vincrinstine (VCR), mitomycin (MMC) and cyclophosphamide (CTX) with MTT and intracellular drug concentration of the transfectants was determined with flow cytometry.RESULTS:In EcoR I and BamH I restriction analysis, the size and the direction of the cloned sequence of Hsp90beta remained what had been designed and the gene constructs were named pcDNA-Hsp90.AH-SGC7901, AH-SGC7901/VCR, AH-HCC7402 and AH-Ec109 cell clones all expressed Hsp90 anti-sense RNA. The expression of Hsp90 was down-regulated in AH-SGC7901, AH-SGC7901/VCR, AH-HCC7402 and AH-Ec109 cell clones. Cell cycle distribution was changed differently. In AH-SGC7901/VCR and AH-Ec109 cells, G(1) phase cells were increased; S phase and G(2) phase cells were decreased as compared with their parental cell lines. In AH-SGC7901 cell, G(1)phase cells were decreased, G(2) phase cells increased and S phase cells were not changed, and in AH-HCC7402 cells G(1), S and G(2) phase cells remained unchanged as compared with their parental cell lines. The sensitivity of AH-SGC7901, AH-SGC7901/VCR, AH-HCC7402 and AH-Ec109 to chemotherapeutic drugs, the sensitivity of AH-SGC7901/VCR to ADR, VCR, MMC and CTX the sensitivity of AH-HCC7402 to ADR and VCR, and the sensitivity of Ec109 to ADR, VCR and CTX all increased as compared with their parental cell lines. The mean fluorescence intensity of ADR in AH-SGC7901, AH-SGC7901/VCR, AH-HCC7402 and AH-Ec109 was also significantly elevated (P 【 0.05).CONCLUSION: Down-regulation of Hsp90 could change cell cycle distribution and increase the drug sensitivity of tumor cells.

Tumor growth inhibition effect of hIL-6 on colon cancer cells transfected with the target gene by retroviral vector

AIM To observe the tumor inhibitory effects bytransfecting IL-6 cDNA into colon cancer cell lineHT-29 with retroviral vector pZIP cDNA.METHODS Human IL-6 gene was reconstructedin retrovirus vector and transfected into incasingcells PA317 by lipofectamine mediated method,the clones of the cells transferred with hlL-6were selected by G418,and targeted HT-29 cellswere infected with the virus granules secretedfrom PA317 and also selected by G418.Test genetranscription and expression level byhybridization,ELISA and MTT assay,etc.Analyze tumor inhibitory effects according to thecell growth curve,plating forming rate andtumorigenicity in nude mice.RESULT Successfully constructed andtransfected recombinant expressing vectorspZIPIL-6 cDNA and got positive transfected celllines.The colon cancer cell line(HT-29 IL-5)transfected with the hlL-5 gene by retroviralvector was established.The log proliferationperiod and the doubling time of this cell line wasbetween 4 to 7 days and 2.5 days according tothe direct cell count,the cell proliferation wasobviously inhibited with MTT assay,the platinginhibitory rate was 50% by plating efficiencytest.When HT-29 IL-6 cells were inoculated intothe nude mice subcutaneously,carcinogenicactivity of the solid tumor was found superior tothe control group and the size of tumor was notsignificantly enlarged.Injection of combinationvirus fluid containing 11.-6 gene intotransplantation tumors could inhibit the growthand development of the tumor.CONCLUSION IL-6 could inhibit the growth andproliferation of colon cancer cells by retroviralvector-mediated transduction.

术中神经电生理监测在单椎板入路选择性神经后根离断术中的作用

目的单椎板入路选择性神经后根离断术(SL-SDR)治疗痉挛性脑瘫完全依赖术中神经电生理监测结果。本研究旨在讨论SL-SDR术中肌电图判读对神经根选择和离断的影响及作用。方法回顾分析2016年5月至2019年3月在上海交通大学附属儿童医院神经外科采用改良神经电生理监测离断方案指导下SL-SDR治疗的痉挛性脑瘫患儿的临床资料,重点关注术中肌电图判读及其与术前临床表现的相关性,以及比较采用不同神经电生理监测离断方案解读术中肌电图时的神经后根选择差异。结果共纳入318例患儿,男性231例、女性87例,平均年龄5.9岁,32例为偏瘫、161例为双侧瘫、125例为四肢瘫。每例患儿有2~8组目标肌群(术前肌张力≥2级)。术中神经电生理监测结果显示,探测的21728枚神经小根[(68.3±8.2)枚/例]中6272枚(28.9%)与肛门括约肌相关,15456枚[(48.6±7.6)枚/例]与下肢肌群相关,其中,神经后根11009枚[(34.6±7.4)枚/例]。共有3370枚[(10.6±4.7)枚/例]符合改良神经后根离断方案,其中3061枚50%部分离断、309枚75%部分离断。术前粗大运动功能分级(GMFCS)分级Ⅰ~Ⅴ级的离断率(部分离断的神经后根数/下肢肌群相关神经后根数)分别为15.8%、22.3%、33.4%、41.8%和45.7%。75%部分离断的离断率随术前GMFCS分级的递增而增加,分别为1.5%、4.8%、8.5%、14.1%和15.2%。术后3周,2068组目标肌群肌张力显著降低[(1.7±0.5)级对(2.7±0.6)级]。术前GMFCS分级Ⅰ和Ⅱ级患儿采用传统离断方案约有20%的病例无法识别任何1枚约20%需离断的神经后根,而随着患儿术前GMFCS分级的递增,传统与改良方案离断的符合率逐步提高,分别为39.5%、41.3%、52.2%、54.1%和62.8%。结论由于SL-SDR完全依赖于术中肌电图判读,使得术中神经电生理监测显得至关重要。改良神经电生理监测离断方案指导下的SL-SDR可以安全、有效治疗痉挛性脑瘫。SL-SDR术中肌电图及其与临床表现相关的预后,可以使临床医师更好地理解此类患儿脊髓神经元环路的运作模式。

单椎板入路选择性神经后根离断术治疗儿童痉挛性偏瘫的可行性和有效性分析

目的传统神经电生理监测离断方案指导下的单椎板入路选择性神经后根离断术(SL-SDR)治疗儿童痉挛性偏瘫仍具有一定挑战性。本研究旨在评估新的改良神经电生理监测离断方案指导下的SL-SDR的可行性和有效性。方法回顾分析2016年5月至2017年10月在上海交通大学附属儿童医院神经外科采用改良神经电生理监测离断方案指导下的SL-SDR治疗,并且术后接受≥12个月强化康复治疗的痉挛性偏瘫患儿的临床和康复资料,重点在于改良方案的术中肌电图判读、手术离断策略及预后。结果共纳入11例患儿,根据新的改良神经电生理监测离断方案,术中共34枚神经后根被离断。经过平均19个月的术后康复治疗,患儿下肢目标肌群肌张力平均下降1.4级,下肢肌力和关节活动度显著改善,术后运动功能明显提高。11例患儿粗大运动功能66项(GMFM-66)评分平均增加10分,6例术前粗大运动功能分级(GMFCS)Ⅱ级患儿中5例术后GMFCS分级降低。部分完成手术前后步态分析的病例显示,11例患儿患侧髋关节、膝关节和踝关节异常运动参数均获得纠正,步态周期中足底压力分布改善明显。无一例出现脑脊液漏、感觉丧失和大小便失禁等手术相关并发症。结论新的改良神经电生理监测离断方案指导下的SL-SDR治疗儿童痉挛性偏瘫是可行、有效的。

Cloning and identification of an angiostatic molecule IP-10/crg-2

AIM To obtain human and murine cDNAsencoding IFN--y inducible protein 10 (lP--10) andcytokine responsive gene--2 (Crg-2).METHODS The encoding genes of lP-10 and Crg2 were amplified by RT--PCR from Cultured humanfibroblast cells and Balb/ c mouse liver treatedby IFN-y and TNF-a, respectively, and clonedinto plasmids of PUC19 and PGEM3Zf(+ ).RESULTS The nucleotide sequences ot theamplified DNA were confirmed by endonucleasesdigestion and sequencing.CONCLUSION Recombinant lP-10/ crg--2 geneclones with 306 hp and 314 hp inserts wereestablished for further research on biologicalactivities and ligands of hiP-10/mCrg--2.

中国碳酸盐岩地区岩溶无机碳汇格局及影响因素

碳酸盐岩化学风化碳汇降低了大气CO_(2)浓度上升和全球变暖的速率,然而,其碳汇通量(CCSF)的估算结果仍存在不确定性,并且气候变化和生态修复对CCSF的贡献率尚不清晰.为此,本文汇编了中国不同流域离子浓度站点数据,并采用经典热力学溶蚀模型,重新评估了中国1991~2020年CCSF的潜力和时空格局,并定量分析了温度(MAT)、降水(MAP)、蒸散发(ET)、土壤水(SM)和归一化植被指数(NDVI)等因子对CCSF的贡献率.结果发现:(1)中国CCSF为22.76t CO_(2)km^(-2)a^(-1),高于全球平均水平(15.77t CO_(2)km^(-2)a^(-1));总量(CCS)为4772.67×10^(4)t CO_(2),以252.98×104km-2的碳酸盐岩面积贡献了全球14.91%的CCS.(2)中国CCSF由东南向西北逐渐减少,其中南方岩溶区、青藏岩溶区和北方岩溶区CCSF分别为33.14、12.93和7.27t CO_(2)km^(-2)a^(-1).(3)1991~2020年中国CCSF整体呈现增加趋势,增长速率为0.16t CO_(2)km^(-2)a^(-1).(4)MAP、MAT、ET、SM和NDVI对CCSF的贡献率分别为63.3%、3.02%、27.5%、3.1%和3.05%,其中降水增加是近30年CCSF上升的主要驱动因子,而蒸发作用的增强抵消了部分降水增加对CCSF的正贡献.总之,本文对中国长时间序列的碳酸盐岩化学风化碳汇量级、格局及影响因素进行系统的量化,这项工作对于国家和全球的碳中和能力诊断和差距分析具有很强的借鉴价值和参考意义.

LRRK2相关帕金森病运动和非运动症状的四年纵向随访研究

帕金森病是一种常见的神经退行性疾病.表现出明显的临床异质性。LRRK2是帕金森病最为常见的致病基因,携带LRRK2致病突变的患者与散发性帕金森病患者临床表现相似。LRRK2风险变异(G2385R,R1628P和S1647T)与亚洲人群发生帕金森病的风险增加有关。以往的研究表明LRRK2突变导致帕金森病患者运动症状进展较快,但未评估其非运动症状的变化特征。为了评估亚洲帕金森病患者中特异性LRRK2风险变异携带者和非携带者的运动和非运动症状的进展特征。