【作者】 席启林；

【导师】 叶章群； 王少刚；

【作者基本信息】 华中科技大学 ， 外科学， 2010， 博士

【摘要】 第一部分靶向大鼠维生素D受体基因miRNA重组腺病毒载体的构建及腺病毒的制备目的：设计、筛选能有效抑制大鼠维生素D受体(VDR)基因表达的miRNA序列,并将miRNA序列与腺病毒载体重组,制备能表达VDR-miRNA的腺病毒。方法：根据大鼠VDR基因序列设计并合成4对miRNA oligo序列(SR-63-1-SR-63-4),退火成双链,将其分别插入到miRNA表达载体pcDNATM6.2-GW/EmGFP-miR中,构建4个miRNA表达质粒,并转化至感受态细胞DH5α。用载体通用引物进行菌落PCR筛选,筛选得到的阳性克隆进行测序,以验证重组克隆中插入片段序列是否与设计的oligo序列一致。将大鼠PC-12细胞VDR克隆至pcDNA3.1质粒构建VDR高表达载体,VDR高表达载体和miRNA干扰质粒共转染293A细胞,Real time q-PCR和Western Blot分别检测共转染干扰效果。将构建成功的靶向大鼠VDR基因的干扰效率最高的miRNA干扰质粒pcDNATM6.2-GW/EmGFP-VDR-miR,通过BP/LR反应,将EmGFP-VDR-miR表达框整合重组到腺病毒载体pAD/CMV/V5-DEST上,测序鉴定。重组腺病毒质粒转染293A细胞,包装收集并检测腺病毒滴度。结果：测序证实pcDNATM6.2-GW/EmGFPmiR干扰质粒中插入了设计的miRNA序列。与NCBI序列比对,pcDNA3.1高表达质粒中插入了大鼠VDR基因序列。Real time q-PCR和Western Blot证实3号干扰质粒(SR-63-3-1)对VDR基因的干扰效果最为明显,在转录水平基因抑制效率达83%。经测序鉴定证实,成功构建了靶向大鼠VDR基因的miRNA腺病毒载体pAD/CMV/V5-GW/EmGFP-VDR-miR,平均滴度为1.44×1011ifu/ml,阴性对照病毒pAd-GFP的平均滴度为7.56×1010ifu/ml。结论：成功构建能在转录和翻译水平有效抑制大鼠VDR基因表达的miRNA腺病毒干扰载体,并制备了高滴度的腺病毒。第二部分腺病毒载体介导维生素D受体基因RNAi对正常大鼠肾上皮细胞钙转运蛋白表达和细胞内游离钙浓度的影响目的：研究维生素D受体(VDR)对正常大鼠肾上皮细胞钙转运蛋白表达的调控作用及对细胞内游离钙离子浓度的影响,以了解VDR在特发性高钙尿症发生中的作用及其机制。方法：利用构建靶向VDR基因RNA干扰的腺病毒载体,转染正常大鼠肾小管上皮细胞(NRK),于转染后48h,72h,96h和120h收集细胞,提取细胞总RNA和蛋白质,使用real time q-PCR和western blot法分别检测干扰前后不同时间点VDR及编码上皮钙转运蛋白基因mRNA和蛋白的表达水平；应用Ca2+荧光探针标记NRK细胞胞浆内游离Ca2+,激光扫描共聚焦显微镜检测干扰前后不同时间点细胞胞浆内游离Ca2+浓度。结果：转染48h后,NRK细胞VDR mRNA和蛋白表达水平开始下降,96h达最低值,与空白对照组比较差异均有统计学意义(P<0.05),而NRK细胞上皮钙转运相关蛋白瞬时感受电位通道V5 (TRPV5)、钙结合蛋白28kd (Calbindin-D28k)和细胞膜钙泵1b (PMCA1b)的mRNA和蛋白表达水平在转染48h后开始下降,72h后与对照组比较差异达统计学意义(P<0.05),96h达最低值。而瞬时感受电位通道V6 (TRPV6)和钠钙交换子1 (NCX1) mRNA和蛋白表达水平在VDR干扰前后不同时间点差异均无统计学意义；同时,抑制NRK细胞VDR基因的表达48h后,胞浆内游离钙离子浓度开始下降,96h降至最低,与基线水平比较,差异有统计学意义(P<0.01),120h后胞浆内钙离子浓度恢复正常。结论：维生素D受体可正性调控正常大鼠肾小管上皮细胞钙转运蛋白TRPV5、Calbindin-D28k和PMCAlb基因的表达,对TRPV6和NCX1基因的表达无明显调控作用,抑制VDR基因的表达可导致NRK细胞胞浆内游离钙离子浓度降低。然而,VDR基因的异常表达在特发性高钙尿症发生中的作用及机制仍需进一步研究。第三部分体内干扰大鼠肾脏维生素D受体基因的表达对大鼠肾组织钙转运蛋白表达及尿钙排泄的影响目的：应用遗传高钙尿结石形成(GHS)大鼠模型,研究肾脏维生素D受体(VDR)异常表达与GHS大鼠尿钙重吸收障碍的相关性,以及发生的分子机制。方法：选取培育的第18代GHS大鼠和正常尿钙SD(NC)大鼠各24只,随机分为6组,平均每组4只。第1-4组采用外科手术方法经肾静脉每侧肾脏分别注入2.5×1010ifu VDR-miRNA干扰腺病毒载体,第5组注入相同剂量的阴性病毒载体作为阴性对照,第6组注入相同体积的生理盐水作为空白对照。第1-4组分别于注入病毒后3、7、14和21天麻醉下取出双肾并处死大鼠,阴性对照组和空白对照组于注射后14天取出双肾并处死大鼠,处死大鼠前2天连续收集2个24小时尿液标本,取肾标本同时留取血清标本。肾组织制作冰冻切片,荧光显微镜观察肾小管上皮病毒感染效率。Real-time q-PCR法和western blot法分别检测肾组织VDR、钙敏感受体(CaSR)和编码上皮钙转运相关蛋白基因的mRNA和蛋白表达水平,后者包括瞬时感受电位通道V5 (TRPV5)、瞬时感受电位通道V6 (TRPV6)、钙结合蛋白9kd (Calbindin-D9k)、钙结合蛋白28kd(Calbindin-D28k)、钠钙交换子1(NCX1)和细胞膜钙泵1b(PMCA1b)。检测尿钙浓度及血清钙、磷、甲状旁腺激素和1,25(OH)2D3水平。结果：GHS大鼠肾组织VDR、Calbindin-D28k和CaSR表达的基础水平比NC大鼠高,而TRPV5、TRPV6、Calbindin-D9k和PMCAlb表达的基础水平比NC大鼠低,差异均有统计学意义(P<0.01)。荧光显微镜观察腺病毒载体成功感染GHS和NC大鼠肾小管上皮细胞,注入VDR-miR RNAi病毒载体后第3天,GHS大鼠肾组织VDR mRNA和蛋白的表达水平较空白对照组和阴性对照组显著降低(P<0.01), VDR基因表达水平在RNAi后第21天达最低值(P<0.01),表明肾组织VDR基因干扰成功,而NC大鼠干扰后VDR的表达水平无显著下降,与空白对照组和阴性对照组比较差异未达统计学意义(P>0.05)。GHS大鼠肾组织VDR基因敲减后导致组织TRPV5、Calbindin-D9k和NCX1基因表达上调,对CaSR和其它上皮钙转运蛋白基因的表达调控作用不明显,而NC大鼠肾组织VDR基因敲减对上述各基因的表达无明显影响。GHS大鼠肾组织VDR基因敲减后第7-21天,24小时尿钙排泄量较干扰前显著增加,差异有统计学意义(P<0.01),NC大鼠24小时尿钙排泄量较干扰前轻微增加,差异未达统计学意义(P>0.05),空白对照组和阴性对照组GHS和NC大鼠24小时尿钙排泄量较干预前轻微增加,但差异无统计学意义(P>0.05)。肾组织VDR基因敲减后对血清钙、磷、甲状旁腺激素和1,25(OH)2D3水平无明显影响。结论：GHS大鼠肾组织VDR基础水平显著高于NC大鼠,而TRPV5基础水平显著低于NC大鼠。GHS大鼠肾脏尿钙重吸收减少可能与肾组织TRPV5基础表达水平下降相关,并且这一过程受VDR调控。肾组织VDR基因的异常表达可能是GHS大鼠产生高钙尿的机制之一。更多还原

【Abstract】 Part1Construction of adenovirus vector-delivered miRNA targeting rat VDR gene and preparation of the adenovirusOBJECTIVE:To design and select the miRNA sequences which can effectively depress the expression of VDR gene, and construct a recombinant adenovirus vector expressing VDR miRNAMATERIALS AND METHODS:Based VDR gene mRNA sequence, four pair of miRNA oligonucletide (SR-63-1-SR-63-4) were designed and synthesized. The oligonucletide were annealed into double strands, then inserted into miRNA expression vector pcDNATM6.2-GW/EmGFPmiR, respectively. The vectors were transformed into competent E.coli DH5αcells. The positive clone was screened with colony polymerase chain reaction and identified by sequencing. VDR cDNA in rat PC-12 cell was cloned into vector pcDNA3.1 to construct the high expression vector containing VDR. The miRNA vector and the high expression vector were co-transfected into 293A cells. Real-time PCR and western blot were used to determine the interference efficacy. The pcDNATM 6.2-GW/EmGFP-VDR-miR vector with the highest RNA interference efficacy was recombined with pAD/CMV/V5-DEST by BP/LR reaction and identified by sequencing. The recombinants were linearized with Pac I and transfected into 293A cells to produce adenovirus. The titer was determined by immune methods.RESULTS:The miRNA oligonucletide sequence was successfully inserted into the plasmid pcDNATM6.2-GW/EmGFPmiR. The third plasmid (SR-63-3) revealed the highest interference efficacy which VDR mRNA knockdown reached 83%. It was confirmed by sequencing that the adenovirus vector was successfully constructed. The mean titer of the positive and negative adenovirus is 1.44×1011ifu/ml and 7.56×1010ifu/ml, respectively。 CONCLUSIONS:The adenovirus vector-delivered miRNA targeting rat VDR was successfully constructed, which could inhabit the mRNA and protein expression of VDR, and the high titer of adenovirus was prepared. Part 2Adenovirus-delivered microRNA targeting VDR reduces intracellular Ca2+ concentration by regulating the express of Ca2+ transport proteins in NRK cellsOBJECTIVE:To determine the effects of vitamin D receptor (VDR) on hypercalciuria and the mechanisms underlying such effects.MATERIALS AND METHODS:The adenovirus vector delivered microRNA targeting rat VDR was constructed. NRK cells (Cellbank, China) were infected with the adenovirus and the cells were collected at 0,48,72,96h after infection. The mRNA and protein levels of VDR and VDR-dependent epithelial Ca2+ transport proteins were detected using real time PCR and western blot assays, respectively. Fluorescent Ca2+ indicator Fluo-4 NW (Fluo-4 NW calcium assay kit, Molecular Probes, Invitrogen, USA) and laser scanning confocal microscope (Olympus, FV500-IX71, Japan) were used to detect the cytosolic free Ca2+ concentration at different time points after infection.RESULTS:The mRNA and protein level of VDR, TRPV5, Calbindin-D28k and PMCAlb in infected NRK cells was significantly lower at 72,96h after infection than that in control cells. No significant difference was found between 2 groups with regard to the mRNA and protein level of TRPV6 and NCX1. Furthermore, VDR knockdown results in a decrease in intracellular Ca2+ concentration in NRK cell lines.CONCLUSIONS:Our study demonstrates that VDR can postively regulate the mRNA and protein expression of TRPV5, Calbindin-D28k and PMCAlb, but not of TRPV6 or NCX1, in NRK cell lines. VDR knockdown results in a decrease in intracellular Ca2+ concentration in NRK cell lines. The effect of the elevated VDR level in kidney on hypercalciuria and the mechanisms underlying need to be further addressed. Part 3Effect of silencing VDR gene in kidney on renal epithelial calcium transporter proteins and urinary calcium excretion in genetic hypercalciuric stone-forming ratsOBJECTIVE:Using genetic hypercalciuric stone-forming (GHS) rats, to address the molecular mechanisms that vitamin D receptor (VDR) in kidney may contributes to decreased renal calcium reabsorption in idiopathic hypercalciuria.MATERIALS AND METHODS:Twenty-four eighteen generation male GHS rats with body weight of 200 to 280 g and Twenty-four Sprague-Dawley male rats with body weight-and age-match were used for these studies. GHS and NC rats were divided into six groups with four of each, respectively. Each kidney for group 1 to 4 rats were injected with 2.5×1010 ifu adenovirus expressing VDR-miRNA via renal venous through surgery. Group 5 received empty adenovirus injection with the same amount through the same transduction methods as negative control. Group 6 received vehicle injection with the same volume as sham-operated control. Groups 1 to 4 were killed 3,7,14 and 21 days after injection, respectively. Group 5 and 6 were killed 14 days after injection. Kidney cortical tissue was removed for subsequent analysis including real time PCR and western blot to quantify VDR, CaSR and epithelial Ca2+ transporters mRNA and protein expression levels, respectively. The latter included TRPV5, TRPV6, Calbindin-D9k, Calbindin-D28k, NCX1 and PMCAlb. Two successive 24h urine were collected before being killed, and blood samples were taken. Urine calcium (UCa) and serum Ca2+, phosphorus and PTH levels were measured on an Abbott Aeroset autoanalyzer. Serum 1,25(OH)2D3 was measured by enzyme-linked immunosorbent assay.RESULTS:Basal levels of epithelial Ca2+ transporters were detected in kidney in GHS and NC rats. The mRNA and protein expression levels in kidney tissues of VDR, Calbindin-D28k, and CaSR were significantly higher in GHS rats than in NC rats. In contrast, the expression levels of TRPV5, TRPV6, Calbindin-D9k, and PMCAlb were significantly lower in GHS rats than in NC rats. Each kidney was injected with adenovirus vector via renal venous through surgery. Parallel GFP expression allowed evaluation of infection efficiency in renal tubular epithelia. It showed the high infection efficiency in kidney in vivo throughout days 3-21. A significant VDR mRNA knockdown with markedly lower VDR protein level was observed after 3 days of infection compared with control and negative groups in GHS(P<0.01). The reduction reached the lowest level after 21 days (P<0.01). This effect was not found in NC rats. VDR knockdown in kidney caused significant increase in renal TRPV5 mRNA expression level in GHS rats compared with control and negative groups. The similar negative regulation of VDR on the renal mRNA expression of NCX1 and Calbindin-D9k was observed in GHS rats. The renal mRNA expression levels of Calbindin-D28k, PMCAlb and CaSR were not altered in response to VDR depletion. Western blot analysis consistently demonstrated the similar regulation in the protein abundance as in the mRNA expression levels. The proteins expression of TRPV5, Calbindin-D9k and NCX1 were negatively regulated, while the renal proteins levels of Calbindin-D28k, PMCAlb and CaSR were unaltered. However, this effect was not detected in NC rats. GHS rats excreted significantly more UCa in days 7-21 after VDR knockdown. Although UCa excretion tended to increase in NC rats and in control and negative groups in GHS ratrs after VDR knockdown or injection of vehicle and negative vector, no significant difference was found. Serum Ca2+, phosphorus, PTH and 1,25(OH)2D3 levels did not significantly changed in response to VDR depletion during different time periods in GHS and NC rats.CONCLUSIONS:Our findings suggest reduced renal Ca reabsorption may be associated with down-regulation of TRPV5 by VDR in GHS rats. This appears to be able to explain the mechanisms that elevated VDR levels contribute to the hypercalciuria. However, the details underlying this effect need to be further elucidated.更多还原