本文選題：CD137L　切入點：共刺激分子　出處：《杭州師范大學》2012年碩士論文　論文類型：學位論文

【摘要】：乙型肝炎病毒(Hepatitis B virus, HBV)持續(xù)感染的主要機理之一是特異性細胞免疫應答不足,而誘導有效細胞免疫的治療性疫苗有望增強抗病毒免疫,可作為HBV感染綜合治療的有效手段之一。因此,有效性、持續(xù)性和安全性兼顧的治療性疫苗是慢性乙肝免疫治療研究的一個重要目標。 目前市售的含鋁佐劑乙肝表面抗原(hepatitis B surface antigen, HBsAg)重組疫苗是有效的預防用疫苗,但由于誘導的細胞免疫應答不足,因而一般不用于抗病毒治療。 CD137/CD137L (4-1BB/4-1BBL)是一對重要的協(xié)同刺激信號分子,對增強和維持免疫應答,尤其是特異性T細胞的擴增、存活和記憶性殺傷等均有極其重要的作用,在抗病毒免疫治療中具有潛在的臨床應用價值。 本研究構建小鼠CD137L的真核表達質粒,觀察該重組質粒和CD137L重組蛋白在小鼠體內對HBsAg重組蛋白和含鋁佐劑乙肝疫苗誘導特異性免疫應答的免疫調節(jié)作用,探討CD137L分子作為乙肝重組疫苗的佐劑以增強抗病毒免疫效應的可能性,為慢性乙型肝炎的治療性疫苗提供新的優(yōu)化思路。 第一部分：小鼠CD137L真核表達質粒的構建及鑒定目的 構建小鼠CD137L的真核表達質粒,并將該質粒體外轉染NIH3T3細胞,檢測其CD137L mRNA和蛋白的表達,為進一步用于動物實驗做好前期準備。方法 1.重組質粒pcD137L的構建 分離BALB/c小鼠脾細胞,體外經(jīng)ConA刺激培養(yǎng)后提取細胞總RNA,通過RT-PCR克隆小鼠CD137L全長cDNA, PCR產物經(jīng)回收和純化后克隆到載體pcDNA3.1(+)上。重組質粒分別經(jīng)PCR、雙酶切和測序鑒定。 2.重組質粒pcD137L的表達及檢測 以脂質體法將重組質粒pcD137L轉染到NIH3T3細胞中,并通過RT-PCR、流式細胞術和免疫熒光法分別檢測轉染細胞中CD137L mRNA和蛋白的表達。結果 1.重組質粒pcD137L的PCR產物擴增出與目的片段相符的單一條帶；雙酶切鑒定得到與目的基因大小一致的片段。 2.測序結果證實所插入片段的核苷酸序列與基因庫中小鼠CD137L基因序列完全一致。 3.經(jīng)RT-PCR從pcD137L轉染細胞中擴增出與目的基因相符的單一條帶。 4. pcD137L轉染細胞經(jīng)PE-抗小鼠CD137L單抗染色后,流式細胞儀檢測到其表面CD137L分子的表達,并在熒光顯微鏡下觀察到這些細胞的胞膜呈現(xiàn)熒光。結論 1.成功構建了小鼠CD137L的真核表達載體(pcD137L). 2.證實重組質粒pcD137L在真核細胞內能成功表達CD137L mRNA和目的蛋白。 第二部分：CD137L重組質粒和CD137L重組蛋白對HBsAg重組疫苗的佐劑作用 目的 分別以CD137L重組質粒(pcD137L)和重組蛋白(rCD137L)與HBsAg重組疫苗共免疫小鼠,探討CD137L分子作為重組乙肝表面抗原(rHBsAg)疫苗的佐劑以增強抗病毒免疫治療作用的可能性。 方法 1.動物分組和免疫： BALB/c小鼠隨機分8個實驗組和5個對照組,每組5只。在0,3,6周各免疫1次,共3次,最后1次免疫后1周處死小鼠。 實驗組為：(1) rHBsAg組(S);(2) rHBsAg+pcDNA3.1(+)組(S+pcD);(3)rHBsAg+pcD137L組(S+pcD137L);(4)rHBsAg+鋁佐劑組(S+Al);(5)rHBsAg+鋁佐劑+pcDNA3.1(+)組(S+Al+pcD);(6) rHBsAg+鉛佐劑+pcD137L組(S+Al+pcD137L);(7) rHBsAg+rCD137L組(S+rCD137L);(8) rHBsAg+鋁佐劑+rCD137L組(S+Al+rCD137LX 對照組為：(1)NS組；(2) pcDNA3.1(+)組(pcD)；(3)鋁佐劑組(A1)；(4) pcD137L組；(5) rCD137L組。 2. ELISA法檢測免疫后小鼠血清IgG, IgGl及IgG2a水平。 3.流式細胞術檢測脾細胞中CD8+T細胞內IFN-y/IL-4表達水平。 4.流式細胞術分析脾細胞中CD44+high釉CD127+CD44+highCD8+T記憶細胞百分比。 5.流式細胞術分析特異性CTL細胞誘導靶細胞凋亡的活性。 結果 1.血清特異性抗體抗-HBs水平檢測 (DpcD137L能促進rHBsAg誘導抗體應答(總IgG、IgG2a和IgGl增高),對含鋁佐劑乙肝疫苗抗體應答無調節(jié)作用。 (2) rCD137L增強rHBsAg誘導Thl型抗體應答(總IgG和IgG2a增高),并可增強含鋁佐劑乙肝疫苗誘導Thl型抗體而下調Th2型抗體(上調IgG2a,下調IgGl). (3)鋁佐劑對rHBsAg誘導抗體有顯著增強作用(總IgG、IgG2a和IgGl增高)。 2. CD8+T細胞內IFN-y/IL-4檢測 (1) pcD137L可促進rHBsAg誘導特異性CD8+T細胞IFN-y的產生(Tcl),并可增強含鋁佐劑乙肝疫苗誘導Tcl反應。 (2) rCD137L對rHBsAg誘導Tcl細胞應答無明顯作用,但對含鋁佐劑乙肝疫苗誘導Tcl細胞免疫應答有顯著促進作用。 (3)鋁佐劑可增強rHBsAg的免疫原性,提高Tcl細胞免疫應答水平。 (4) pcD137L和rCD137L對rHBsAg或含鋁佐劑rHBsAg刺激特異性CD8+T細胞分泌IL-4均無明顯調節(jié)作用。 3.脾細胞CD8+記憶T細胞分析 (1) pcD137L促進rHBsAg產生CD44+high和CD127+CD44+highCD8+T記憶細胞,但對含鋁佐劑乙肝疫苗誘導CD8+記憶T細胞無明顯作用。 (2) rCD137L可促進rHBsAg誘導CD8+記憶T細胞形成,并可增強含鋁佐劑乙肝疫苗產生CD8+記憶T細胞。 (3)鋁佐劑促進rHBsAg疫苗誘導CD8+記憶T細胞數(shù)。 4.特異性CTL誘導靶細胞凋亡的活性分析 (1) rCD137L增強rHBsAg及含鋁佐劑rHBsAg誘導特異性CTL應答。 (2)鋁佐劑可增強rHBsAg疫苗的CTL活性。 以上差異均有統(tǒng)計學意義。結論 1. CD137L重組質粒增強HBsAg重組疫苗在小鼠體內誘導的抗體應答、Tcl細胞應答及CD8+記憶T細胞形成,但對含鋁佐劑乙肝疫苗無明顯免疫增強作用。 2.CD137L重組蛋白顯著促進含鋁佐劑HBsAg重組疫苗在小鼠體內產生IgG2a (Thl型)抗體反應和以分泌IFN-y為特征的Tc1(I型)細胞免疫,增強其特異性CTL活性及CD8+記憶T細胞形成,可能是市售乙肝疫苗由預防用制劑轉為治療性疫苗的有效佐劑。
[Abstract]:Hepatitis B virus (Hepatitis B, virus, HBV) is one of the main mechanism of persistent infection is the specific cellular immune response and induce cellular immune deficiency, effective therapeutic vaccine could enhance antiviral immunity, can be used as one of the effective means of comprehensive treatment of HBV infection. Therefore, the validity, continuity and security of both therapeutic vaccine is an important target on chronic hepatitis B immune therapy.
Currently, the commercially available recombinant hepatitis B surface antigen (HBsAg) recombinant vaccine is an effective preventive vaccine, but it is generally not used for antiviral therapy because of its insufficient cellular immune response.
CD137/CD137L (4-1BB/4-1BBL) is one of the important costimulatory signal molecule, maintain and enhance immune response, especially the amplification of specific T cell and the effect of survival and mnemonic killer has the extremely important and has potential clinical value in antiviral therapy.
The construction of eukaryotic expression plasmid of CD137L mice, observe the immune regulation effect of recombinant plasmid and CD137L recombinant protein in mice by recombinant HBsAg protein and aluminum adjuvant hepatitis B vaccine induced specific immune response, the possibility of CD137L as a molecular adjuvant of recombinant HB vaccine to enhance the antiviral immune effect, provide new optimization ideas for the treatment of chronic hepatitis B vaccine.
Part 1: Construction and identification of CD137L eukaryotic expression plasmids in mice
The eukaryotic expression plasmid of mouse CD137L was constructed, and the plasmid was transfected into NIH3T3 cells in vitro, and the expression of CD137L mRNA and protein was detected.
Construction of 1. recombinant plasmid pcD137L
The spleen cells from BALB/c mice were isolated, cultured in vitro, and the total RNA was extracted after ConA stimulation. The full length cDNA of mouse CD137L was cloned by RT-PCR, and PCR products were cloned into vector pcDNA3.1 (+) by recycling and purification. The recombinant plasmids were identified by PCR, double enzyme digestion and sequencing.
Expression and detection of 2. recombinant plasmid pcD137L
The recombinant plasmid pcD137L was transfected into NIH3T3 cells by liposome method, and the expression of CD137L mRNA and protein in transfected cells was detected by RT-PCR, flow cytometry and immunofluorescence.
1. the PCR product of recombinant plasmid pcD137L amplified a single band that was consistent with the target fragment, and double enzyme digestion was used to identify the fragments that were in accordance with the size of the target genes.
The results of 2. sequencing confirmed that the nucleotide sequence of the inserted fragment was exactly the same as the mouse CD137L gene sequence in the gene pool.
3. the single band that was consistent with the target gene was amplified by RT-PCR from the transfected cells from pcD137L.
4. pcD137L transfected cells were stained with PE- anti mouse CD137L monoclonal antibody. The expression of CD137L on their surface was detected by flow cytometry, and the fluorescence of these cells was observed under fluorescence microscope.
1. the eukaryotic expression vector (pcD137L) of mouse CD137L was successfully constructed.
2. it was confirmed that the recombinant plasmid pcD137L could successfully express CD137L mRNA and target protein in eukaryotic cells.
The second part: the effect of CD137L recombinant plasmid and CD137L recombinant protein on the adjuvant of HBsAg recombinant vaccine
objective
Mice were immunized with recombinant plasmid CD137L (pcD137L) and recombinant protein (rCD137L) and recombinant HBsAg vaccine respectively. The possibility of CD137L as a adjuvant of recombinant hepatitis B surface antigen (rHBsAg) vaccine was explored to enhance the effect of antiviral immunotherapy.
Method
1. animal groups and immunization:
BALB/c mice were randomly divided into 8 experimental groups and 5 control groups, with 5 rats in each group. 1 times each were immunized at 0,3,6 weeks, and the mice were killed for 1 weeks after the last 1 immunizations.
The experimental group: (1) rHBsAg group (S); (2) rHBsAg+pcDNA3.1 (+) group (S+pcD); (3) rHBsAg+pcD137L group (S+pcD137L); (4) rHBsAg+ aluminum adjuvant group (S+Al); (5) rHBsAg+ aluminum adjuvant +pcDNA3.1 (+) group (S+Al+pcD); (6) rHBsAg+ lead adjuvant + pcD137L group (S+Al+pcD137L); (7) rHBsAg+rCD137L group (S+rCD137L); (8) +rCD137L group (S+Al+rCD137LX rHBsAg+ aluminum adjuvant
The control group was: (1) NS group; (2) pcDNA3.1 (+) group (pcD); (3) aluminum adjuvant group (A1); (4) pcD137L group; (5) rCD137L group.
The serum levels of IgG, IgGl and IgG2a after immunization were detected by 2. ELISA method.
The expression of IFN-y/IL-4 in CD8+T cells in spleen cells was detected by 3. flow cytometry.
4. flow cytometry was used to analyze the percentage of CD44+high enamel CD127+CD44+highCD8+T memory cells in splenocytes.
5. flow cytometry was used to analyze the activity of specific CTL cells to induce the apoptosis of the target cells.
Result
Detection of anti -HBs level of 1. serum specific antibodies
(DpcD137L can promote rHBsAg induced antibody response (the increase of total IgG, IgG2a and IgGl), and has no regulatory effect on the antibody response of HBV vaccine containing aluminum adjuvant.
(2) rCD137L enhanced rHBsAg induced Thl type antibody response (increased total IgG and IgG2a), and increased the Thl antibody induced by hepatitis B vaccine with aluminum adjuvant, and down regulated Th2 antibody (up IgG2a and down IgGl).
(3) the aluminum adjuvant enhanced the antibody induced by rHBsAg (total IgG, IgG2a and IgGl).
Detection of IFN-y/IL-4 in 2. CD8+T cells
(1) pcD137L can induce rHBsAg to induce the production of IFN-y in specific CD8+T cells (Tcl), and enhance the Tcl reaction induced by the hepatitis B vaccine containing aluminum adjuvant.
(2) rCD137L has no obvious effect on rHBsAg induced Tcl cell response, but it can significantly promote the immune response of Tcl cells induced by hepatitis B vaccine containing aluminum adjuvant.
(3) aluminum adjuvant can enhance the immunogenicity of rHBsAg and improve the level of Tcl cell immune response.
(4) both pcD137L and rCD137L had no obvious regulatory effect on rHBsAg or aluminum containing adjuvant rHBsAg stimulated by specific CD8+T cells to secrete IL-4.
Analysis of CD8+ memory T cells in 3. splenocytes
(1) pcD137L promoted rHBsAg to produce CD44+high and CD127+CD44+highCD8+T memory cells, but had no obvious effect on CD8+ memory T cells induced by the hepatitis B vaccine containing aluminum adjuvant.
(2) rCD137L can induce rHBsAg to induce the formation of CD8+ memory T cells and enhance the CD8+ memory T cells produced by the vaccine of hepatitis B containing adjuvant hepatitis B.
(3) aluminum adjuvant promoted the rHBsAg vaccine to induce the number of CD8+ memory T cells.
Analysis of the activity of 4. specific CTL induced apoptosis in target cells
(1) rCD137L enhanced rHBsAg and aluminum containing adjuvant rHBsAg induced specific CTL responses.
(2) the aluminum adjuvant can enhance the CTL activity of rHBsAg vaccine.
The above differences were statistically significant.
1. CD137L recombinant plasmid enhanced the antibody response induced by HBsAg recombinant vaccine in mice, Tcl cell response and CD8+ memory T cell formation, but had no significant immune enhancing effect on hepatitis B vaccine containing aluminum adjuvant.
The recombinant 2.CD137L protein significantly promote aluminum adjuvant HBsAg recombinant vaccine IgG2a in mice (Thl) antibody reaction and to secrete IFN-y as the characteristics of the Tc1 (I) cell immunity, enhance the specific activity of CTL and CD8+ T memory cell formation, may be sold by the prevention of hepatitis B vaccine preparations for effective adjuvant therapy vaccines.

【學位授予單位】：杭州師范大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R392.11

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相關期刊論文 前1條

1 江虹;黃茵;史麗云;吳煒;蔡玲斐;賈紅宇;鐘石根;;CD137L在HBsAg DNA疫苗誘導小鼠細胞免疫應答中的佐劑效應[J];浙江大學學報(醫(yī)學版);2010年04期

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