人黑素瘤细胞A375 MIA/CD-RAP基因的克隆及其鉴定

目的为了研究人黑素瘤M IA/CD-RAP基因的生物学功能,克隆M IA/CD-RAP的cDNA,构建表达载体后,对其进行酶切和DNA测序鉴定。方法用RT-PCR从培养的人黑素瘤细胞A375中扩增M IA/CD-RAP cDNA,将其克隆到载体pcDNA3.1-MycH is上,经酶切及PCR扩增鉴定和测序。结果酶切电泳和DNA测序结果表明,成功地克隆了人黑素瘤M IA/CD-RAP cDNA。结论人黑素瘤M IA/CD-RAP cDNA的克隆,可为进一步研究其生物学功能奠定基础。     

皮肤科学基础

诱导皮肤免疫抑制和表没食子儿茶素没食子酸酯光保护作用;中波紫外线对皮肤免疫系统影响的某些研究进展（综述）;全反式维甲酸和阿达帕林对中波紫外线诱导的melan-α黑素细胞黑素合成的影响;人黑素瘤细胞A375MIA／CD-RAP基因启动子的克隆及其鉴定;过氧化氢对黑素细胞生物学作用的研究……[第一段]     

黑素瘤MIA启动子介导的CD/TK双自杀基因重组腺病毒系统选择性杀伤黑素瘤细胞

目的:探讨黑素瘤抑制蛋白(melanoma inhibitory activity,MIA)启动子介导的CD/TK双自杀基因重组腺病毒系统,对黑素瘤细胞A375的选择性杀伤作用。方法:构建腺病毒穿梭质粒pAdrMIA-CD/TK,在293细胞内包装、扩增、纯化后,体外转染人表皮黑素细胞HeMa-LP、黑素瘤细胞A375和人宫颈癌细胞HeLa,RT-PCR检测基因CD和TK片段,加入前体药物5-氟胞嘧啶(5-FC)和/或丙氧鸟苷(GCV),用MTT法测定该体系对细胞株的杀伤效应。结果:制备的病毒滴度为1×1011pfu/L,用100m.o.i.重组腺病毒感染细胞后,发现目的基因CD和TK片段可在黑素瘤细胞中表达,转染目的基因的黑素瘤细胞A375对5-FC和GCV具有一定的敏感性,细胞存活率较对照组显著下降(P<0.05),联合应用两种药物对A375细胞的杀伤作用较单用一种显著增强(P<0.05)。对人表皮黑素细胞HEMa-LP和人宫颈癌细胞HeLa无显著杀伤作用。结论:构建的黑素瘤MIA启动子介导的CD/TK双自杀基因重组腺病毒系统,在体外可选择性杀伤黑素瘤细胞A375。     

耐药淋球菌差异DNA消减文库的构建及初步筛选

目的 克隆和研究耐药性淋球菌相关基因。方法 从耐药淋球菌株RSM292C4和淋球菌标准参考菌株WHO-A细胞中提取DNA,经RsaⅠ酶切后,应用抑制性消减杂交技术构建耐药性淋球菌差异DNA消减文库并进行初步筛选。结果 耐药淋球菌差异DNA消减文库经扩增获得约2500个白色克隆,随机挑取96个白色克隆,进行PCR扩增鉴定,发现克隆中插有100~600bp大小的片段。分别以RsaI酶切样本和参照DNA为探针,与随机挑取的96个白色菌落质粒作点杂交,有5个克隆仅能与样本DNA探针杂交,而不能与参照DNA探针杂交,表明它们为RSM292C4基因组DNA所特有,可能代表某些淋球菌新基因。对这5个克隆插入的DNA片段进行测序,经送基因库和淋球菌基因组部分测序基因库检索分析,发现5个片段均为未知新序列。结论 淋球菌耐药菌株DNA消减文库的构建为进一步筛选、克隆淋球菌耐药的新基因奠定了基础。     

信号肽序列修饰的单纯疱疹病毒-2多表位复合DNA疫苗构建

目的:构建经糖蛋白gD信号肽修饰的单纯疱疹病毒2型(HSV-2)糖蛋白gD、gB、gC和早期表达蛋白ICP 27的多表位复合DNA疫苗.方法:国内HSV-2野生株经测序与PCR鉴定后利用PCR技术从其基因组中扩增出HSV-2 ICP27 377-459、gD146-179、gD223-306、gB529-606、gC247-282、gD1-77 6个基因片段,PCR鉴定后按先后顺序,采用多基因片段一步酶切连接法获得HV融合基因片段,经pGEMT载体克隆后定向插入真核表达质粒pcDNA3.1载体中,构建出重组真核表达质粒HV-pcDNA3.1,并对其进行酶切分析及测序鉴定.PCR扩增gD146-179信号肽片段,进一步酶切连接构建重组质粒gDs-HV-his-pcDNA3.1,并对其进行测序鉴定.结果:酶切重组质粒HV-pcDNA3.1,电泳可见两条带,分别为融合基因HV(1.3 kb)和线性质粒pcDNA3.1(5.4 kb).测序结果表明,克隆基因插入方向正确,重组质粒gDs-HV-his-pcDNA3.1与GenBank HSV-2中相关序列同源性达99.9%.结论:成功构建了经糖蛋白gD信号肽修饰的HSV-2多表位复合DNA疫苗.     

皮肤科学基础

20091785 人β-Catenin-EGFP融合表达慢病毒载体构建及在毛囊干细胞中的表达/杨鹏高(上海交大医学院三院),胡孝辉,高丰厚…∥上海交通大学报(医学版).-2009,29(9).-1030～1034提取人血管内皮细胞总RNA,RT-PCR扩增获得β-连环蛋白(β-Catenin)基因全部序列,采用TA克隆技术获取基因亚克隆PVCm-T-β-Catenin,Age I酶切连接PGE-FV载体构建β-Catenin融合增强型绿色荧光蛋白(EGFP)共表达慢病毒载体质粒PGC-FV-β-Catenin-EG-FP。质粒转化感受态细菌,筛选阳性克隆,经RT-PCR及测序鉴定正确后转染FT     

MIA基因-RNAi对人黑素瘤A375细胞增殖的影响

目的探讨RNA干扰技术抑制恶性黑素瘤细胞株A375MIA基因表达后,对A375细胞增殖的影响。方法构建针对人黑素瘤A375细胞(MIA)基因3个不同靶序列的RNAi真核表达载体pLTE-hMIA-RNAi,用磷酸钙细胞转染法把构建的载体分别转染HEK293细胞,采用Western技术和RT-PCR技术分别检测MIA基因蛋白的表达和mRNA水平。选择对MIA基因表达抑制作用最强的RNAi载体转染A375细胞后,用细胞计数检测细胞增殖的变化。结果构建的3个RNAi质粒和对照组相比,pLTE-hMIA-RNAi352质粒对MIA的表达减少量>80%,hMIA的mRNA水平下降约83%,与对照组比较差异有显著性意义。而pLTE-hMIA-RNAi203、pLTE-hMIA-RNAi343质粒对MIA的表达及mRNA水平的影响,与对照组比较差异无显著性意义。转染pLTE-hMIA-RNAi352质粒的A375细胞增殖能力较对照组显著下降。结论构建的针对人MIA基因352位靶序列的RNAi质粒pLTE-hMIA-RNAi352可显著抑制MIA的表达及A375细胞增殖。     

CD147siRNA表达载体的构建与鉴定

目的 构建CD147siRNA表达载体,分析CD147在皮肤肿瘤浸润和转移中的作用机制。方法 根据基因库上的CD147 cDNA序列,设计并合成两端含有酶切位点的64个碱基的寡核苷酸链。寡核苷酸链退火后用T4DNA连接酶连接到线性化的pSUPER质粒中,并对重组质粒(命名为pSUPER/CD147 siRNA)进行酶切、PCR及测序鉴定。结果 PCR扩增片段与预期结果相符,双酶切证实CD147 siRNA表达载体克隆构建成功,插入片段测序结果与合成的siRNA结果一致。结论 成功构建CD147 siRNA表达载体,CD147可为治疗肿瘤开辟新途径。     

黑素瘤抑制性活性因子在黑素瘤不同阶段及基底细胞癌中的表达

目的：探讨黑素瘤抑制性活性因子（MIA）在黑素瘤及基底细胞癌中的表达及其作用。方法：应用sP免疫组化技术检测38份黑素瘤石蜡标本、35份基底细胞癌石蜡标本以及32份色素痣石蜡标本中MIA的表达水平。结果：MIA在所有色素痣以及基底细胞癌中均呈阴性表达，而在原位黑素瘤、侵袭性黑素瘤、有淋巴结转移的黑素瘤、无淋巴结转移的黑素瘤阳性表达率分别为21．4％、91．6％、94．1％和42．8％。结论：MIA在黑素瘤的发生发展中起重要作用，MIA有可能成为临床诊断、治疗黑素瘤的靶点。     

短发卡RNA对人黑素瘤细胞BRAF基因的沉默作用

目的构建BRAF基因序列特异性的短发卡RNA(shRNA)质粒载体,检测其对人黑素瘤细胞BRAF基因的干扰作用。方法设计2条BRAF基因序列特异性的shRNA编码序列和1条非特异性阴性对照序列,插入质粒pGenesil-1,双酶切和测序鉴定。用构建成功的3条质粒转染人黑素瘤细胞株A375和M14,分别采用RT-PCR和蛋白印迹检测其BRAF基因的mRNA和蛋白表达。结果所设计的shRNA序列被正确插入质粒pGenesil-1。非特异性对照质粒neg对黑素瘤细胞BRAF基因表达不产生干扰,2条序列特异性质粒braf1和braf2抑制了BRAF基因mRNA和蛋白的表达,其中braf1干扰效果最为明显,最高抑制率可达90%。结论质粒介导的shRNA可成功干扰人黑素瘤细胞BRAF基因的表达,其抑制时间可持续至少1个月。     

Impact of lymph node metastases on serum level of melanoma inhibitory activity in stage III melanoma patients

Melanoma patients in stage III have a considerable recurrence rate. The 10-year survival in this stage depends on the number and size of affected nodes. Currently, there is no optimal serum marker for early detection of relapse available. The goal of the study was to assess the utility of melanoma inhibitory activity (MIA) serum marker in the follow up and primary diagnosis of stage III melanoma patients. One hundred and thirty-eight melanoma patients in stage III at time of primary diagnosis were analyzed at time of primary diagnosis and during periodical routine follow up both for serum MIA using an enzyme-linked immunosorbent assay and for serum lactate dehydrogenase (LDH). Results were correlated with the positivity of the sentinel lymph node (SLN) and the number of lymph node metastases in the completion lymph node dissection at time of primary diagnosis. During follow up, the overall survival time was assessed using the Kaplan-Meier method in terms of elevated MIA (>12 ng/mL) values. Regarding SLN status, significant differences of MIA values (P = 0.024) and LDH (P = 0.007) were found, both within the normal cut-off. Having lymph node metastases in the completion lymph node dissection, significantly higher MIA values (12.55 ng/mL [±0.48], P < 0.0001) were found. In patients with three or more tumor-positive nodes, MIA values were significantly higher when compared to patients with one or two affected nodes (P = 0.024). In the routine follow-up, stage III patients with an MIA value of more than 12 ng/mL had a five times higher risk for developing recurrences (P < 0.0001). Patients with relapsing disease had a significantly (P < 0.0001) higher mean MIA value (13.76 ng/mL) compared to patients without relapse (7.52 ng/mL). The MIA serum marker can be helpful in patients undergoing lymph node dissection. Furthermore, during follow up, patients showing relapsing diseases can have an elevated MIA value.     

白介素22在皮肤黑素瘤细胞中的表达

目的：探索白介素（IL）-22和IL-22相关的细胞因子受体在皮肤黑素瘤细胞中的生物学活性及其相互间的关系。方法：通过荧光素酶试验对信号转导子和催化子3（STAT3）活性的分析，观察IL-22与其受体（IL-22R）之间的相互关系。通过基因转染技术和PCR检测，分析IL-22与IL-22R结合蛋白（IL-22BP）的相互关系。结果：IL-22与IL-10R、IL-22R结合后能够诱导信号转导子和STAT3活化。基因转染实验显示IL-22BP具有抑制IL-22活性的功能。结论：IL-22、IL-10R、IL-22R和IL-22BP在黑素瘤细胞中具有相互调节的作用。这种调节作用在皮肤黑素瘤的治疗中有一定的研究价值。     

依巴斯汀通过抑制AKT/mTOR通路诱导人黑素瘤细胞自噬

目的：研究依巴斯汀对人黑素瘤细胞自噬的影响及机制。方法：体外培养人黑素瘤细胞A375和M14,采用CCK-8增殖实验检测细胞活力,并计算IC50;利用mCherry-EGFP-LC3B双荧光指示系统检测自噬流;采用Western blot验证自噬相关蛋白LC3,Beclin1及信号通路蛋白的表达。结果：依巴斯汀可显著抑制人黑素瘤细胞的活力;依巴斯汀明显诱导人黑素瘤细胞中自噬小体和自噬溶酶体的产生;依巴斯汀显著上调人黑素瘤细胞中LC3-Ⅱ/Ⅰ的比值以及Beclin1的表达,同时抑制AKT/mTOR信号通路的活化,降低p-AKT/AKT和p-mTOR/mTOR。结论：依巴斯汀通过抑制AKT/mTOR通路诱导人黑素瘤细胞自噬的发生。     

Efficient and selective tumor cell lysis and induction of apoptosis in melanoma cells by a conditional replication‐competent CD95L adenovirus

Please cite this paper as: Efficient and selective tumor cell lysis and induction of apoptosis in melanoma cells by a conditional replication‐competent CD95L adenovirus. Experimental Dermatology 2010; 19 : e56–e66. Abstract: The high mortality of melanoma demands the development of new strategies, and gene therapy may be considered provided improvements in efficacy and selectivity. Overexpression of the death ligand CD95L/FasL has been shown in previous studies as highly effective for apoptosis induction in melanoma cells. For efficient and selective targeting of melanoma, a conditional replication‐competent adenoviral vector was constructed (Ad5‐FFE‐02), which drives CD95L expression by a tetracycline‐inducible promoter. For restricting its replication to melanoma cells, the adenoviral E1A gene is controlled by a tyrosinase‐derived promoter. Furthermore, adenoviral E1B was deleted and a mutated E1A was used to preferentially support replication in tumor cells. Proving its high selectivity and efficiency, strong expression of E1A and doxycycline‐dependent induction of CD95L were characteristic for tyrosinase‐positive melanoma cells after Ad5‐FFE‐02 transduction, whereas absent in non‐melanoma cell lines. Importantly, Ad5‐FFE‐02‐mediated cell lysis was restricted to melanoma cells, and induction of apoptosis was found only in tyrosinase and CD95 expressing cells. Finally, the combination of adenoviral replication and CD95L‐mediated apoptosis resulted in an enhanced repression of melanoma cell growth. This new adenoviral vector may provide a basis for an efficient targeting of melanoma.