摘要
研究背景：
良性前列腺增生(benign prostate hyperplasia, BPH)是一种渐进性的病理过程。在过去的50年里,有关BPH的病因已有大量的研究证实,其中雄激素与其受体在前列腺增生的发生和发展中起着重要作用,但它不是导致BPH的决定原因,也没有一种理论能够确定其中的因果关系。尽管人们早就发现在增生的前列腺组织标本中免疫细胞浸润与炎症反应的存在,但这一现象却被大多数临床与病理医生所忽视,更低估了炎症免疫过程在BPH发生、发展中的关键作用。炎症免疫过程在BPH病因和症状进展中可能起着关键作用。病理组织学研究发现：前列腺免疫学改变通常先于BPH的组织学改变。在BPH的发展中,免疫炎症和间质细胞互相作用与影响,使BPH长期存在慢性炎症免疫应答过程。前列腺免疫学已发展成为BPH病因研究的一个新领域,人们开始思考与研究炎症免疫与前列腺增生、进展是否存在因果关系,BPH组织中白细胞增多的原因与特征,在炎症免疫与BPH反应过程中,BPH组织细胞、免疫细胞、促炎症细胞因子三者之间在前列腺分子免疫网络中的调节效应与生物学功能,性激素与炎症免疫在BPH发生、发展过程中的作用,已成为前列腺免疫学研究的热点问题。
组织学前列腺炎(histological prostatitis)的病因目前尚不是很清楚。其主要的局限在于组织标本的获取困难以及诊断标准的不统一,导致对其的研究一直未获进展,其对前列腺组织生理功能的影响以及与各类前列腺临床症状发生的相关性也缺乏进一步的研究。因此有必要对组织学前列腺炎的病因学以及其对前列腺症状的影响进行深入的研究。目前对组织学前列腺炎标本的获取主要来自前列腺增生患者,因此对组织学前列腺炎的研究通常兼顾对前列腺增生的研究。
维生素D即1,25-(OH)2D3,在体内必须通过维生素D受体(vitamin D receptor, VDR)发生作用。VDR是亲核蛋白,属类固醇激素／甲状腺激素受体超家族成员,是介导1,25-(OH)2D3发挥生物学效应的核内大分子。VDR是核受体超家族的成员之一,存在于靶细胞核内。人类VDR基因位于12q13-14上,全长约78000bp,由11个外显子和11个内含子组成。VDR分为核受体(nVDR)和膜受体(mVDR)2类。nVDR影响基因表达,控制相应蛋白质的合成,mVDR则主要参与钙磷平衡的维持。人体几乎所有细胞均有VDR的存在。1,25-(OH)2D3在体内具有多种生物学功能,如调控免疫应答、控制细胞增殖和分化、体内矿物质平衡等,其功能主要是通过VDR介导的。VDR具有广泛的生物学效应,包括维持体内血清钙和磷的稳定,调节细胞增殖、分化及免疫调节功能等。其与1,25-(OH)2D3结合,可改变VDR的转录效率。调节钙磷代谢是VDR的主要功能。近年来发现其在对机体的免疫功能亦有重要的调节作用。VDR与多种疾病的发生有相关性,已成为近年来医学研究的热点。
单核苷酸多态性(single nucleotide polymorphism, SNP)是人类基因组中最常见、分布最广泛的DNA多态性类型,大约每1000bp就存在一个SNP。是人类基因组中物理图谱的理想遗传标记,能满足对代谢、生长和疾病相关基因的定位。被认为是继限制性片段长度多态性和微卫星多态性之后的第三代遗传标记。在人类基因组中大约有300万个SNP。SNP在群体中的发生频率不小于1%。SNP所表现的多态性只涉及到单个碱基的变异,这种变异可由单个碱基的转换(transition),即一种嘌呤替换成另一种嘌呤,或一种嘧啶替换成另一种嘧啶；或颠换(transversion),即嘌呤与嘧啶之间的互换所引起；也可由碱基的插入或缺失所致。转换与颠换的比例为2：1。SNP有如下几个特点：(1)密度高。SNP是目前为止人类基因组中分布最广泛、数量最多的的DNA多态型,大约90%的人类遗传变异是SNP。而STR在整个基因组中约15kb才有一个STR位点。(2)片段短。SNP基因座的片段更短,更易进行PCR扩增,并且产物的长度不到100bp,这与300～400bp的STRs相比能够更好地适用于降解的DNA样本。(3)遗传稳定性强。STR在基因组中存在不稳定和密度相对较低,SNP具有更高的遗传稳定性,可以用于群体遗传学的研究。(4)易于高通量、自动化分析。SNP大都表现为二等位基因标记,在检测时只需要通过简单的“+／-”或“有／无”来进行基因分型,易于进行分型和确定基因频率,有利于各实验室基因判型的标准化和质量控制,适于快速、高通量检测和自动化分析。SNP是目前人类基因组的一个研究热点,在遗传作图、群体遗传学、疾病预防诊断治疗、癌症等方面具有不可估量的影响。
已有研究结果表明在不同人群中VDR基因上存在有SNP的差异。由于存在SNP种族的差异性,从而导致不同种族人群对某些疾病有遗传易感染性。VDR基因启动子上存在Fok Ⅰ位点F/f单核苷酸多态性,FokⅠ位点多态性造成了VDR mRNA转录和表达水平上的差异,进而造成VDR活性不同,而VDR活性的改变会导致其许多生理功能发生变化。其中与组织学前列腺炎有关系的功能之
为免疫调节。目前已经发现的VDR基因上存在的与疾病发生有关的基因多态性位点有Bsm Ⅰ、Taq Ⅰ、Fok Ⅰ等酶切位点,其中FokⅠ位于启动子上,启动子结构的改变对因的表达会产生重要的调节作用。已有的研究发现VDR基因多态性与某些慢性炎症性疾病的发生有一定关系。王振华等的研究发现VDR基因多态性分布差异与小儿反复呼吸道感染有一定的相关性。他们选取的是Bsm-Ⅰ多态性位点,发现反复呼吸道感染的患儿B等位基因频率有增高的趋势。张立等对124例慢性牙周炎患者和91例健康对照者分析后发现,VDR上的Taq Ⅰ位点多态性中,t等位基因可提高中国汉族人群患早发性牙周炎的风险。李升等运用Meta分析对TaqⅠ基因多态性与慢性牙周炎易感性进行了研究,发现388名慢性牙周炎患者和355名对照者中TaqⅠ基因多态性与慢性牙周炎的易感性无关。因此,VDR基因多态性与非感染性炎症性疾病的关系值得进一步研究。
在分析和研究SNP的诸多方法中,通过聚合酶链反应—限制性片断长度多态性方式(polymerase china reaction-restriction fragment length polymorphism, PCR-RFLP)为目前运用较多且十分成熟的方法。其基本原理为：首先利用DNA提取技术分离和纯化目的基因片段,然后运用PCR技术对目的基因片段进行特异性扩增,再利用针对特殊酶切位点的生物酶进行目的基因片段的酶切,最后对酶切后的片段进行电泳,判读其基因型。其中PCR技术经过多年的发展,已经成为研究基因组学相当成熟的实验平台,用于扩增目的基因。而RFLP技术也在不断进步和发展,为SNP相关研究提供了有力的技术支持。
目的和意义：
1.本研究通过PCR-RFLP方法检测前列腺增生合并组织学前列腺炎和单纯良性前列腺增生患者VDR基因启动子上存在的FokⅠ位点SNP基因型,分析VDR基因启动子上存在的FokⅠ位点SNP不同的基因型与前列腺增生合并组织学前列腺炎发生之间的相关性。探讨组织学前列腺炎的发生有无遗传易感性,寻找组织学前列腺炎的遗传性病因。
2.探讨VDR基因启动子上存在的FokⅠ位点SNP是否为组织学前列腺炎发生的风险因素之一,为指导组织学前列腺炎的一级预防提供依据。
3.通过分析VDR基因启动子上存在的FokⅠ位点SNP与组织学前列腺炎严重程度分型之间的关系,为指导组织学前列腺炎的病理诊断分型以及指导临床治疗提供一定的参考。
4.通过基因测序方法检验PCR-RFLP方法分析SNP基因的准确性和可重复性。为开展SNP的分析提供实验指导。
方法：
收集2008年8月至2011年11月在广州市红十字会医院泌尿外科治疗的前列腺增生合并组织学前列腺炎患者169例(炎症组),术后组织病理检查示前列腺增生,慢性前列腺炎,平均年龄73.9±2.7岁。同时收集156例单纯前列腺增生患者(对照组),病理检查示前列腺增生,平均年龄73.56±2.68岁。两组患者标本均为行经尿道前列腺电切(TURP)术后获得。取得标本后,常规行10%的中性福尔马林浸泡。行常规病理组织学检查。并收集两组患者相关基本临床资料,PSA, IPSS评分,前列腺体积,尿流率以及前列腺增生药物治疗情况等。
通过聚合酶链反应-限制性片段长度多态性(Polymerase chain reaction-restriction fragment length polymorphism, PCR-RFLP)检测研究对象VDR基因启动子上存在的Fok Ⅰ位点SNP基因型：1.从NCBI的gene bank中检索VDR基因的相关资料,获得其基因序列,2.采用Genefisher软件设计PCR引物,3.利用TIANamp Genomic DNA Kit试剂盒提取目的基因DNA片段,4.在PCR仪上进行目的基因的扩增,5.鉴定扩增后的目的基因片段,6.对扩增后目的基因片段进行特异性酶切,7.收集酶切后的片段进行琼脂糖凝胶电泳,8.对电泳结果进行判读。9.通过基因测序方法对酶切结果进行分析,评估PCR-RFLP的准确性。
运用常规病理方法检查单纯良性前列腺增生以及前列腺增生合并组织学前列腺炎的发生情况,并对组织学前列腺炎的严重程度进行分型,以此为依据进行研究对象的分组。
统计学处理：
所有数据采用SPSS13.0统计软件分析。良性前列腺增生合并组织学前列腺炎和单纯前列腺增生患者的年龄,PSA, IPSS评分,前列腺体积,尿流率等相关临床资料对比采用t检验。根据Hardy-weinberg定律,采用直接计算法计算两组基因型和等位基因频率的实际数和预期数,了解各基因型在人群中遗传平衡的符合程度,采用χ2检验。比较两组基因型以及等位基因频率的差异性,采用χ2检验。两两比较用分割的μ~2检验。比较不同严重程度的组织学前列腺炎之间基因型以及等位基因频率的差异性,采用χ2检验。采用多元Logistic回归法分析VDR基因启动子上存在的FokⅠ位点SNP不同的基因型与BPH合并组织学前列腺炎发生风险之间的关系。通过逐步回归对控制年龄等因素后的风险进行评估。P<0.05说明差异有统计学意义,P<0.01说明差异有显著统计学意义。
研究内容和过程：
1.本研究采用病例对照研究方法,制定良性前列腺增生合并组织学前列腺炎以及单纯前列腺增生的纳入以及排除标准,选定研究对象,2.用TIANampGenomic DNA Kit试剂盒(深圳晶美公司提供)提取169例前列腺增生合并组织学前列腺炎患者以及156例单纯前列腺增生的目的基因片段,3.运用PCR方法扩增目的基因片段,4.通过RFLP方法分析研究对象VDR基因启动子FokⅠ位点SNP基因型,5.通过基因测序方法对酶切结果进行分析,评估PCR-RFLP实验的准确性,6.运用常规病理检查方法确诊单纯良性前列腺增生以及前列腺增生合并组织学前列腺炎,同时对组织学前列腺炎的严重程度进行分类,7.通过直接计算以及统计学方法进行两组研究对象的Hardy-weinberg平衡检验,分析观察对象的群体代表性,8.通过统计学软件分析两组观察对象基本临床资料差异性有无统计学意义,9.运用统计学方法分析VDR基因启动子FokⅠ位点SNP与BPH合并组织学前列腺炎之间的相关性,并分析该SNP与组织学前列腺炎严重程度之间的相关性,10.运用单因素回归分析法,分析VDR基因启动子上存在的FokⅠ位点SNP是否为组织学前列腺炎发生的风险因素。
结果：
1.对良性前列腺增生合并组织学前列腺炎以及单纯前列腺增生患者VDR基因FokⅠ位点SNP基因型分布的实际数和预期数进行直接计算,再通过χ2检验,结果显示实际数与预期数差异无统计学意义(P>0.05),符合Hardy-Weinberg定律,在遗传学上说明该人群资料具有代表性。
2.对两组患者的相关临床资料进行比较用t检验。结果显示两组患者平均年龄无显著差异(P=0.118),炎症组PSA水平(ng/ml)显著高于对照组(P=0.001)；两组患者术前血常规以及尿常规检查未见感染,肝,肾功能检查未见异常。国际前列腺症状(I-PSS)评分：炎症组为15.4±2.5分,对照组为15.3±2.32分,两组之间的差异无统计学意义(P=0.744)。两组患者术前行经腹部B超测量前列腺体积,用左右、前后、上下三组径线(毫米,mm)表示,炎症组前列腺体积(毫升,ml)=0.52×(前列腺三径的乘积)显著大于对照组(P=0.027),术后前列腺切除重量(克,g)无统计学意义(P=0.423)炎症组有35例术前无急性尿潴留,常规行尿流率检查,对照组有40例行尿流率(ml/s)检查,两组其余患者均已停留尿管,两组间已行的尿流率的差异无统计学意义(P=0.626)。
3.对两组患者VDR基因启动子FokⅠ位点SNP基因型进行比较,采用χ2检验。结果显示ff基因型在炎症组为37%(62/169),对照组为25%(39/156),该基因型在两组间的分布差异有统计学意义(P<0.05),而FF和Ff基因型在两组之间的分布为：炎症组：25%(43/169)；对照组：35%(55/156)和38%(64/169)；40%(62/156),差异无统计学显著性意义(P>0.05)。F基因频率在炎症组为44%(150/338),对照组为55%(172/312),f基因频率在炎症组为56%(188/338),对照组为：45%(140/312),两组之间的差异有统计学意义(P<0.05)。
4.本研究所选人群中重度组织学前列腺炎三种基因型的分布不符合Hardy-Weinberg定律,故该类型在本研究中不具有人群代表性。而轻度以及中度组织学前列腺炎三种基因型的分布符合Hardy-Weinberg定律。故分析三种基因型在轻度和中度组织学前列腺炎之间的差异性。结果显示FF、Ff以及ff基因型在轻度炎症组与中度炎症之间的分布为：32%(25/79),23%(12/52)；46%(36/79),40%(21/52)；22%(18/79),37%(19/52),差异无统计学显著性意义(P>0.05)。F基因频率在轻度炎症组和中度炎症组分别为54%(86/158),43%(45/104),f基因频率在两组的分别为46%(72/158),57%(59/104),两组之间的差异无统计学意义(P>0.05)。
5.通过基因测序方法对PCR-RFLP的实验结果进行分析,随机抽取三种基因型各10例进行基因测序,发现基因型符合实验前的基因排列。验证了PCR-RFLP实验的准确性和可重复性。
6.以VDR基因启动子FokⅠ位点F/f单核苷酸多态性的基因型为自变量,以BPH合并组织学前列腺炎作为因变量,进行BPH合并组织学前列腺炎危险因素Logistic回归分析。结果显示,在控制了年龄等因素后,FokⅠ多态性是前列腺增生合并组织学前列腺炎发生的风险因素(P<0.05)。其中ff基因型相对于FF基因型的OR值为2.03(0.279-0.866)其中Ff基因型相对于FF基因型的OR值为1.32(0.446～1.287),其中ff基因型相对于Ff基因型的OR值为1.54(0.382～1.105)。
结论
1.VDR基因启动子FokⅠ位点SNP中ff基因型以及f基因等位频率在前列腺增生合并组织学前列腺炎和单纯前列腺增生患者中的分布差异有统计学意义,说明ff基因型与组织学前列腺炎的发生有相关性。VDR基因启动子FokⅠ位点SNP作为一种遗传标记,说明组织学前列腺炎的发生有人群遗传易感性。从而为组织学前列腺炎的病理分型以及临床治疗提供一定的指导。
2.通过单因素Logistic回归分析VDR基因启动子FokⅠ位点SNP与前列腺增生合并组织学前列腺炎发生之间的关系,发现VDR基因启动子FokⅠ位点SNP是组织学前列腺炎发生的独立危险因素。为从基因水平治疗组织学前列腺炎提供参考。
3.VDR基因启动子FokⅠ位点SNP不同基因型的分布与组织学前列腺炎严重程度的差异无统计学意义,需进行更大样本量的研究揭示该SNP位点与组织学前列腺炎的病理分型的相关性。
4.组织学前列腺炎对PSA以及前列腺体积有一定的影响。
Background:
Benign prostatic hyperplasia (BPH) is a kind of incremental pathological process. In the last50years, there has been a large number of relevant research about cause of BPH. Androgen and its receptor play an important role in occurrence and development of BPH. But it is not the only cause of BPH. It is not a theory to be able to determine the causality. Although it has been found immune cells infiltration and inflammation in existence in tissue sample of BPH, but this phenomenon was ignored by most clinical and pathological doctor, underestimated the inflammatory process play a key role in occurrence and development of BPH. Inflammatory immune process causes and symptoms of BPH progress may play a key role. The pathological and histological study found that prostate immunology change usually ahead of the histologic BPH. In the development of BPH, immune inflammation and stromal cells function and influence each other, make BPH long-standing in chronic inflammatory immune response process. Prostate immunology has become a new field of study in cause of BPH, people began to research inflammatory and BPH. The cause and characteristics of the increase of white blood cells in BPH. In the process of immune with BPH, some reserches focus on BPH organization cells, immune cells, promote inflammation cell factors in the regulating effect and biological function inflammatory reaction process in prostate molecules immune network. Sex hormones and immune inflammation in occurrence, development of BPH has become a hot issue of prostate immunology. Histological prostatitis (histological prostatitis) cause is not yet clear. The main limitation of tissue samples for difficulty lies in the diagnostic criteria of unity and not, lead to its research has not won its development, physiological function of prostate tissue, the influence of the prostate with all kinds of clinical symptoms occur correlations also lack of further research. So it is necessary to histological prostatitis etiology and its influence of the prostate symptoms of further research. At present the specimen obtain histological prostatitis mainly from patients with prostate hyperplasia, so the histologic prostatitis research usually pay attention to the prostatic hyperplasia.
Vitamin D is1,25-(OH)2D3, it plays a part in the body through the vitamin D receptor (VDR). VDR is one member of the steroid hormone/thyroid hormone receptor superfamily, which is the nuclear macromolecules mediated1,25-(OH)2D3play the biological effects. VDR presents in the cell nucleus, it is one member of nuclear receptor superfamily. Human VDR gene is located in12q13-14, stretches approximately78000bp, by11explicit the son, and11of introns. VDR divided into nuclear receptor (nVDR) and membrane receptor (mVDR)2class. NVDR affects gene expression, control the corresponding protein synthesis, mVDR is mainly involved in the maintenance of calcium phosphate balance. The human body cells are almost all the existence of VDR.1,25-(OH)2D3has a variety of biological function in the body, such as regulation immune response, control cell proliferation and differentiation, body mineral balance, its main functions are mediated by VDR.
VDR has extensive biological effects, including in maintaining the stability of the serum calcium and phosphorus, regulating cell proliferation, differentiation and immune adjustment function, etc. Its and1,25-(OH)2D3combination can change VDR transcription efficiency. Regulating calcium phosphate metabolism is the main function of VDR. In recent years, it was be found that it also have been an important regulatory role in immune function. VDR and a variety of the happening of the disease have relevance, has become the hot spot of the medical research in recent years.
Single nucleotide polymorphism (SNP) is the most common in the human genome, the most widespread DNA polymorphisms type. It has a SNP in every1000bp. It is the human genome physical maps of ideal genetic markers, can meet the metabolism, growth and the orientation of disease genes. It is be considered to be the third generation of genetic markers after the restriction fragment length polymorphism and microsatellite polymorphism. There are about3million SNP in the human genome. The frequency of the SNP is not less than1%in population. SNP showed polymorphism involves only to a single base of variations that can be made of a single base conversion (transition), namely a purine replaced by another purine, or a pyrimidine into another replace a pyrimidine; or transversion, namely purine and pyrimidine exchange between the site. It also a result of insert or missing of the base. The proportion of transtion and transversion is2:1. SNP there are several characteristics:(1) high density. SNP is by far the most widespread in the human genome, the most number of DNA polymorphisms type, about90%of the human genetic variation is the SNP. And in the whole genome STR about15KB have a STR site.(2) short clips. The SNP loci blocks more short, easier for PCR amplification, and the length of the product is less than100bp, this and300～400bp than STRs can better apply to degradation DNA samples.(3) the genetic stability strong. STR in existing in the genome instability and density is lower, the SNP has higher genetic stability, and can be used in the study of population genetics.(4) easy to high throughput, automatic analysis. Most of the performance for the second class SNP a gene markers and the test only need through the simple "+/-" genotyped, easy for classification and determine the gene frequency, be helpful for the laboratory to the standardization of gene type and quality control, and is suitable for fast, high throughput detection and automatic analysis.In theory, the SNP are generally considered to be a bi-allelic markers. In the genome SNP screening often needs only (+/-)analysis. Now SNP is one focus of the human genome research. It has inestimable influence in common diseases in the genetic susceptibility, disease prevention, diagnosis and treatment for cancer.
Existing research results show that different people in the genes exist VDR SNP differences. Due to the differences of SNP race there, leading to different ethnic groups for some disease has a genetic easy infectious.There is f/F single nucleotide polymorphisms in VDR gene promoter Fok I site. Fok I site polymorphism caused the differences of VDR mRNA in the level transcription and expression. It cause the different of VDR activity. The change of VDR activity can lead to many of its physiological function change. The histological prostatitis have relationship for one of the functions is immune regulation.
At present, have been found on the genes of the existence of VDR and disease of the genetic polymorphism Bsm I, Taq I, Fok I were cut site, which is located in the Fok I promoter, start to change because of the substructure of expression will be produced important regulatory role. Of the existing research found that VDR genetic polymorphism with some chronic inflammatory the happening of the disease have particular concern. WangZhenHua found that the gene polymorphism VDR distribution difference and pediatric repeatedly has certain correlation respiratory infections. They selected is Bsm I polymorphism site, respiratory infection of children repeatedly found B allele frequency have higher trend. ZhangLi such as of124cases of chronic periodontitis patients and91cases of healthy controls were analysis found that, on Taq I sites of VDR polymorphism, t allele can improve the han people in China with periodontitis praecox the risk. Dr such as using Meta analysis to Taq I with chronic periodontitis susceptibility gene polymorphism, found that the388chronic periodontitis patients and355controls were Taq I with chronic periodontitis the susceptibility of business. Therefore, VDR genetic polymorphism and noninfectious inflammatory disease relationship deserves further study.
In the analysis and research of many of the SNP method, through the polymerase chain reaction-restrictive fragment length polymorphism means (PCR-RFLP) for now uses more mature and very method. The basic principle is:first use DNA extraction separation and purification purpose gene fragments, and then use PCR technology on purpose gene fragments specificity amplification, and then cut for special enzymes sites on purpose gene fragments of biological enzyme cut, finally to cut the enzyme after fragments electrophoresis, reading their genotype. One PCR technology after years of development, has become a mature genomics of experimental platform, for purpose gene amplification. And also in progress RFLP technology and development for the SNP related research provides a powerful technical support.
Objective:
The purpose of this study is analysis the relationship VDR gene promoter Fok I sites SNP with BPH complicated histologic prostatitis by researching VDR gene promoter F Explore VDR gene promoter are Fok I sites with histological prostatitis SNP happened between the risk, Fok I SNP genotype. Discusses whether the histologic prostatitis happened genetic predisposition.
Through the analysis of VDR gene promoter are Fok I SNP and histological prostatitis locus of points, the relationship between the guidance for histologic prostatitis pathological points and the clinical treatment of guidance to provide certain reference.
It is explored the heredity factors in the cause of histologic prostatitis by this artical. The result may provide some clues in further studying development process of BPH.
Methods:
Collecting August2008to November in Guangzhou Red Cross hospital uropoiesis surgical treatment of prostate hyperplasia merger histologic prostatitis patients169cases (inflammation group), postoperative histopathologic examination reealed prostate hyperplasia, chronic prostatitis, mean age73.9±2.7years old. At the same time data of156patients with simple prostatic hyperplasia (control group), pathological examination reealed prostate hyperplasia, mean age73.56±2.68years old. Two groups of patients are through the urethra prostate specimen cutting (TURP) postoperative gain. After get specimens, the conventional line of10%in darfur-marin soaked. Do conventional colonoscopy. Two groups of patients, and collect relevant basic clinical material, PSA, IPSS score, prostate gland volume, urine flow rate, prostatic hyperplasia drug treatment, etc.
VDR gene promoter Fok I SNP genotype are protected by polymerase chain reaction-restriction fragment length polymorphism(PCR-RFLP). The diagnosis of BPH or BPH complicated histologic prostatitis by conventional pathological methods. The relationship between risk of VDR gene promoter Fok I sites SNP with BPH complicated histologic prostatitis was analysed by Logistic regression model. The difference of two groups were analysed by statistical methods.1. The gene bank from NCBI in the relevant information retrieval VDR genes, and obtain the genetic sequence,2. Genefisher software design using PCR primer,3. Use TIANamp Genomic DNA Kit Kit extraction purpose gene segments of DNA,4. In the PCR on the instrument purpose gene amplification,5. Identify the purpose of pieces after amplification fund6. After the expansion of purpose gene fragments specificity enzyme cut,7. Collect enzyme after cutting the fragments agarose gel electrophoresis,8. The results of electrophoretic interpretation. Using conventional pathological methods to check and pure BPH BPH merger histologic prostatitis of occurrence, and on the basis of the research object group.
The statistical treatment:
All data using SPSS13.0statistical software analysis. Benign prostatic hyperplasia histological prostatitis and simplicity with prostate hyperplasia of the patient's age, PSA, IPSS score, prostate gland volume, urine flow rate, and other relevant clinical data comparison inspection by t. According to the Hardy-weinberg's law, adopt direct calculation method for calculation of two groups of genetic type and allele frequency of the actual number and the expected number, understand each type gene in the crowd of genetic balance with degree, the χ2inspection. Compare two sets of genetic type and allele frequency differences, using χ2inspection. Comparing different degree of histological prostatitis genotype and allele frequency differences, using x2inspection. The single factor Logistic regression analysis VDR gene promoter are Fok I SNP different genes with BPH histologic type with risk of prostate inflammation of the correlation between. P<0.05instructions are statistically significant differences, P<0.01that there is a significant difference of statistical significance.
The research content and process:
The study was made by control studies. Two groups of subjects were divided by into and exclusion standard.
The DNA of169patients with BPH complicated histologic prostatitis and156with BPH were abstracted from blood respectively.
Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was used in determining VDR genotypes.
BPH and BPH complicated histologic prostatitis were diagnosed by using conventional pathology.
The differences of two groups object basic clinical data were analysed by statistical software.
The object group representative and Hardy-weinberg balance inspection were calculated by the direct calculation method and statistics method.
The relationship of VDR gene promoter Fok I sites SNP with BPH complicated histologic prostatitis was analysed by statistical methods.
Results:
For benign prostatic hyperplasia histological prostatitis and merger pure prostatic hyperplasia VDR gene Fok I sites with SNP genotype distribution of the actual number and the expected number directly computing, again through the χ2inspection, the results show the actual number and the expected number was not statistically significant difference (P>0.05), meet Hardy-Weinberg's law, in this population genetics explain representative data.In two groups of patients with relevant clinical data of comparison with t test. The results suggest that both groups of patients are representative data.
In two groups of patients with relevant clinical data of comparison with t test. The results suggest that both groups of patients, average age no significant difference (P=0.118), inflammation group PSA levels (ng/ml) significantly higher than those in the control group (P=0.001). Two groups of patients preoperative routine blood and urine check has not seen the infection, liver, kidney check has not seen the exception. International prostate symptoms (I-PSS) score:inflammation for15.4±2.5points group, compared with15.3±2.32points, the difference between the two groups was statistically significant (P=0.744). Two groups of patients through B ultrasonic measure before the abdomen prostate olume, and use around, front and back, and three groups, diameter line (mm) said, inflammation group prostate olume (ml)=0.52×(prostate is the product of the three diameter) significant higher than control group (P=0.027), postoperative prostate removed weight (g) was not statistically significant (P=0.423) inflammation group has35patients preoperative no acute urinary retention, routine urine flow rate do check, the control group40routine urine flow rate (ml/s) check, two groups of the rest of the patients were already stay urinary catheter, between the two groups have done urine flow rate of the difference was not statistically significant (P=0.626).
Two groups of patients for VDR gene promoter Fok Ⅰ SNP compared gene loci type, the χ2inspection. Results showed that genetic type of inflammation in ff was37%(62/169), the control group was25%(39/156), the genetic type in the distribution difference between the two groups was statistically significant (P<0.05), and ff and ff genotype between the two groups for:the distribution of inflammation group:25%(43/169); The control group:35%(55/156) and38%(64/169);40%(62/156), the difference was not statistically significant meaning (P>0.05). F gene frequency in inflammation group is44%(150/338), the control group was55%(172/312), F gene frequency in inflammation group is56%(188/338), the control group for:45%(140/312), the difference between the two groups was statistically significant (P<0.05).
The research selected the crowd moderate to severe histologic prostatitis three genotype distribution is not in conformity with the Hardy-Weinberg's law, so this type in this study does not have the crowd representative. And mild and moderate histologic prostatitis three genotype distribution meet Hardy-Weinberg's law. It analyzes three genotype in mild to moderate histologic prostatitis differences between the. Results showed that FF, FF and genotype mild inflammation in FF group and moderate inflammation of distribution between for:32%(25/79),23%(12/52);46%(36/79),40%(21/52);22%(18/79),37%(19/52), the difference was not statistically significant meaning (P>0.05). F gene frequency in mild inflammation group and moderate inflammatory group were54%(86/158),43%(45/104), F gene frequency in both groups were46%(72/158),57%(59/104), the difference between the two groups was statistically significant (P>0.05).
Through the gene sequencing method to the experimental result of the PCR-RFLP analysis, three kinds of random genetic type,10cases of the gene sequencing, discovered genes with before the experiment of gene content. Verify the accuracy of PCR-RFLP experiment and repeatability.
To VDR gene promoter Fok I site F/f SNP genotype as the independent variable and merger with BPH histological prostatitis as the dependent variable, the merger BPH histological prostatitis risk factors Logistic regression analysis. The results showed that in the control factors such as age, Fok I polymorphism is prostate hyperplasia merger histologic prostatitis happen risk factors (P<0.05). One genotype relative to ff ff genotype OR value of2.03(0.279～0.866) which ff genotype relative to ff genotype OR value of1.32(0.446～1.287), including ff genotype relative to ff genotype OR value of1.54(0.382～1.105).
Conclusion:
1. VDR gene promoter Fok I sites in genotype and the SNP ff gene allele frequency in f prostate hyperplasia histological prostatitis and pure with the distribution of patients with prostate hyperplasia difference was statistically significant, explain ff genotype and histological prostatitis occurs with correlation. VDR gene promoter Fok I SNP sites as a genetic markers that histologic prostatitis occurs with the crowd of genetic predisposition.
2. Through the single factor Logistic regression analysis VDR gene promoter Fok I sites and prostatic hyperplasia with SNP histological prostatitis happen, the relationship between the found VDR gene promoter Fok I SNP sites is histologically prostatitis happened independent risk factor. From the genetic level for treatment of prostatitis histologically to provide the reference.
3. VDR gene promoter Fok I sites different genotypes of SNP the distribution and histological prostatitis serious degree of difference was statistically significant, need to undertake more research reveals the sample SNP sites and histological prostatitis pathological points the correlation of type, thus for pathology classification and clinical treatments with certain guidance.
4. Histologic prostatitis affect the PSA and prostate voleume to a certain extent.
引文
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