本文選題：基因型　切入點(diǎn)：慢性乙型肝炎　出處：《新鄉(xiāng)醫(yī)學(xué)院》2014年碩士論文

【摘要】：背景 慢性乙型肝炎(chronic hepatitis B, CHB)的發(fā)病機(jī)制目前普遍認(rèn)為與免疫損傷有關(guān)。干擾素用于CHB治療時(shí),具有抗病毒和免疫調(diào)節(jié)的雙重作用,病人的感染狀態(tài)和基礎(chǔ)免疫狀況可能決定其治療的療效和穩(wěn)定性,但其機(jī)制尚不清楚。T細(xì)胞受體(T cell receptor, TCR)互補(bǔ)決定區(qū)3(complementarity determining region3,CDR3)譜型分析技術(shù)是一項(xiàng)很好的反映機(jī)體細(xì)胞免疫功能的新方法。本研究采用該技術(shù)分析CHB患者基線狀態(tài)的細(xì)胞免疫功能情況,并探討其與干擾素治療早期病毒學(xué)應(yīng)答的關(guān)系。 目的 通過對(duì)入組患者的血清HBV基因型進(jìn)行檢測(cè),初步了解本地區(qū)HBV基因型的分布情況；利用實(shí)時(shí)熒光定量PCR溶解曲線分析技術(shù)了解不同基因型CHB患者基線狀態(tài)的T細(xì)胞克隆性增生情況,探討基線狀態(tài)的細(xì)胞免疫功能與干擾素抗病毒治療近期療效的關(guān)系,以了解基礎(chǔ)免疫狀況對(duì)干擾素抗病毒近期療效的評(píng)估作用,指導(dǎo)臨床治療。 方法 (1)選取2013年4月至2013年10月在新鄉(xiāng)醫(yī)學(xué)院第一附屬醫(yī)院感染內(nèi)科住院接受干擾素-α治療的CHB患者23例,治療前留取外周靜脈血5m1,肝素鈉抗凝,采用密度梯度離心法分離血清和外周血單個(gè)核細(xì)胞(peripheral blood mononuclearcell,PBMC)；(2)采用S基因測(cè)序法檢測(cè)HBV基因型：根據(jù)文獻(xiàn)設(shè)計(jì)并合成所用引物,提取血清中的HBV-DNA用于PCR擴(kuò)增模板,擴(kuò)增產(chǎn)物進(jìn)行瓊脂糖凝膠電泳和S基因區(qū)測(cè)序,測(cè)序結(jié)果用DNASTAR處理,并與GenBank/EMBL/DDBJ數(shù)據(jù)庫中的A-G7種HBV基因型參考序列進(jìn)行比較,進(jìn)化樹分析用MEGA5.1軟件,確定出基因型；(3)提取PBMC總RNA,逆轉(zhuǎn)錄為cDNA,設(shè)計(jì)并合成TCRβ鏈24個(gè)可變區(qū)基因(BV)家族上游引物,并在p鏈恒定基因區(qū)(BC)設(shè)計(jì)一條共同的無熒光標(biāo)記的下游引物。采用熒光定量PCR擴(kuò)增TCR各BV家族CDR3區(qū)基因,溶解曲線分析各樣本PCR產(chǎn)物形成的24個(gè)TCR BV家族溶解曲線譜型圖,溶解后的產(chǎn)物重新短暫擴(kuò)增后,擴(kuò)增產(chǎn)物進(jìn)行1.5%的瓊脂糖凝膠電泳分析,了解各BV家族的表達(dá)情況；(4)同時(shí)觀察基線及治療3個(gè)月HBV-DNA、肝功能水平變化;(5)統(tǒng)計(jì)學(xué)分析應(yīng)用PSS16.0軟件進(jìn)行,計(jì)量資料以均數(shù)±標(biāo)準(zhǔn)差(x±s)或中位數(shù)/四分位數(shù)間距(M/QR)表示,對(duì)數(shù)據(jù)進(jìn)行獨(dú)立樣本t檢驗(yàn)、配對(duì)樣本比較Wilcoxon符號(hào)秩檢驗(yàn)、兩獨(dú)立樣本比較的Wilcoxon符號(hào)秩檢驗(yàn),P0.05為差異有統(tǒng)計(jì)學(xué)意義。 結(jié)果 (1)入組的23例CHB患者中,C2基因型21例(91.3%),B2基因型1例(4.34%),D基因型1例(4.34%)。 (2)23例CHB患者基線時(shí)外周血TCR CDR3各BV家族譜系在溶解曲線譜型圖上均表現(xiàn)不同程度的譜型偏移,出現(xiàn)單峰、寡峰及偏峰圖,多數(shù)家族表現(xiàn)為多峰圖,小部分家族由于表達(dá)頻率極低或缺失表現(xiàn)為無峰圖。對(duì)照的GAPDH表現(xiàn)為明顯的單峰圖形,陰性對(duì)照無熔解曲線峰。擴(kuò)增產(chǎn)物在1.5%的瓊脂糖凝膠上電泳,結(jié)果顯示多數(shù)BV家族均有表達(dá),在預(yù)測(cè)位置處顯示一條模糊的條帶,部分家族表達(dá)增強(qiáng)或缺失,顯示一條清晰條帶或條帶缺失。GAPDH對(duì)照呈現(xiàn)單條帶,陰性對(duì)照無條帶。 (3)23例CHB患者干擾素-a治療3個(gè)月與基線比較,血清谷丙轉(zhuǎn)氨酶(alanine aminotransferase, ALT)、谷草轉(zhuǎn)氨酶(glutamic-oxaloacetic transaminase, AST/GOT)水平及HBV-DNA載量均明顯下降(P0.01)；根據(jù)譜型分析結(jié)果,將21例C基因型CHB患者分為TCR CDR3高譜型偏移率組(譜型偏移率≥35%)和低譜型偏移率組(譜型偏移率35%)。TCR CDR3高譜型偏移率組與低譜型偏移率組比較,干擾素治療過程中HBV-DNA載量下降幅度更明顯(P0.05), ALT、AST下降水平間差異均無顯著性(P0.05)。 結(jié)論 本地區(qū)HBV基因型以C2基因型為主,極少數(shù)為B型或D型；不同基因型的CHB患者基線狀態(tài)外周血T淋巴細(xì)胞存在不同程度的克隆性增生；C基因型CHB患者中基線外周血T細(xì)胞表現(xiàn)為高克隆性增生的感染者在干擾素抗病毒治療過程中HBV-DNA載量下降幅度更明顯,此可能與干擾素治療較好的近期療效相關(guān)。
[Abstract]:Background

The pathogenesis of chronic hepatitis B and chronic hepatitis B is generally considered to be related to immune injury . When the interferon is used in the treatment of chronic hepatitis B , it has the double effect of anti - virus and immunoregulation . The infection state and the basal immune status of the patient may determine the therapeutic effect and stability of the patients . The technique is used to analyze the cellular immune function of the patients with chronic hepatitis B . The relationship between the immune function and the early virological response is discussed .

Purpose

The distribution of HBV genotypes in the region was preliminarily determined by the detection of HBV genotypes in patients with enrolled patients .
Using real - time fluorescence quantitative PCR dissolution curve analysis technique to understand the T cell clonal expansion of different genotypes and patients with different genotypes , the relationship between the cellular immune function of baseline status and the short - term efficacy of interferon antiviral therapy was discussed to understand the effect of basal immune status on the short - term efficacy of interferon antiviral therapy , and to guide clinical treatment .

method

( 1 ) From April 2013 to October 2013 , 23 patients who received interferon - 偽 were treated with interferon - 偽 in the First Affiliated Hospital of Xinxiang Medical College from April 2013 to October 2013 . Peripheral blood mononuclear cells ( PBMC ) were isolated from peripheral blood mononuclear cells ( PBMC ) by density gradient centrifugation .
( 2 ) detecting HBV genotypes by using S gene sequencing method : designing and synthesizing the primer according to the document , extracting HBV - DNA in the serum for PCR amplification template , carrying out agarose gel electrophoresis and S gene region sequencing , sequencing the amplified products by DNASTAR , and comparing with the reference sequence of the A - G7 type HBV genotype in the GenBank / EMBL / DDBJ database , and determining the genotype by using the MEGA5.1 software ;
( 3 ) The total RNA of PBMC was extracted , and the upstream primer of TCR 尾 chain 24 variable region genes ( BV ) was designed and synthesized .
( 4 ) The baseline and 3 months of HBV - DNA and liver function changes were observed at the same time ; ( 5 ) Statistical analysis was performed using PSS16 . 0 software . The measurement data was expressed by mean 鹵 standard deviation ( x 鹵 s ) or median / quartile range ( M / QR ) . The data was subjected to independent sample t test . The paired samples were compared with Wilcoxon signed rank test , and the Wilcoxon signed rank test between two independent samples was statistically significant .

Results

( 1 ) Of the 23 patients , the C2 genotype was 21 ( 91.3 % ) , B2 genotype was 1 ( 4.34 % ) , genotype D was 1 ( 4.34 % ) .

( 2 ) Twenty - three patients with hepatitis B showed a different degree of spectral shift on the spectrum of dissolution profiles . There were single peaks , few peaks and partial peaks . Most of the families showed multiple peaks . The results showed that most of the families of BV had a single peak pattern , and the negative control showed no melting curve . The results showed that most BV families were expressed and a clear band or band deletion was displayed at the predicted position .

( 3 ) The levels of alanine aminotransferase ( ALT ) , aspartate - oxaloacetic transaminase ( AST / GOT ) and HBV - DNA were significantly decreased ( P0.01 ) .
According to the results of the spectral analysis , 21 patients with genotype C were divided into three groups with high spectral type ( 鈮，

本文編號(hào)：1713835