出版年:

研究生:

鄢搖培

研究生(英文姓名):

Yao-Pei Yan

論文名稱:

存在於台灣族群具有阻絕HIV-1感染的特異基因與細胞因子之探討

英文論文名稱:

The genes and cellular factors that block HIV-1 infection in Taiwanese

指導教授:

蘇益仁 ; 黎慶

指導教授(英文姓名):

Ih-Jen Su ; Ching Li

學位類別:

碩士

校院名稱:

國立成功大學　

系所名稱:

微生物暨免疫學研究所

學號:

S46851055

學年度:

87

語文別:

英文

論文頁數:

65

關鍵詞:

台灣族群 ; HIV-1 ; CCR5 ; HLA

英文關鍵詞:

HIV-1 coreceptor ; CCR5 m303 ; CCR5 delta32 ;
beta-chemokines ; HLA ; Taiwanese ; MIP-1 ;
RANTES

被引用次數:

0

[ 摘要 ]

HIV-1是感染人類的反轉錄病毒，它利用表面的醣蛋白與細胞上的受體作用並經與細胞膜融合後進入宿主細胞。HIV-1感染細胞最初的受體是CD4分子，而所利用的副受體(coreceptor)分子是與病毒株的細胞屬性有關。若HIV-1感染的細胞是以巨噬細胞(macrophage)及原始的T細胞(primary T cells)為主，則副受體為b-chemokine的受體CCR5 (稱為M-tropic或是R5病毒株)；若病毒株感染的細胞以原始的T細胞及transformed T細胞為主，則副受體為a-chemokine的受體CXCR4 (稱為T-tropic或是X4病毒株)。以前的研究指出，HIV-1的副受體與其相對的嵌合分子(ligand)在HIV-1的致病機制扮演很重要的角色。譬如帶有成對同體的(homozygous) 32個核甘酸CCR5缺失基因型(稱為CCR5D32)，或是體內有高濃度b-chemokines (MIP-1a,、MIP-1b、RANTES)與interleukin (IL)-16的高加索人就不會被HIV-1感染。雖然CCR5 D32以及高表現量的b-chemokines與IL-16能防止HIV-1感染，但是這些基因及細胞因子尚未在中國人的種族做有系統的調查。因此，我們首先在台灣地區調查了369位不同社會背景的族群，包括有接觸到HIV-1而不被感染的人、HIV-1高危險群的公娼、HIV-1帶原者及一般正常人的CCR5基因型，然而我們並沒有發現任何D32的存在。另外有報導指出CCR5有單一突變的對偶基因(allele)稱為m303，能保護高加索人不被M-tropic HIV-1病毒株感染，因此我們更進一步在台灣族群調查了CCR5 m303的基因型。如同CCR5D32的結果，我們分析的個體中並沒有發現任何m303的存在(n=316)。這些結果證明了CCR5D32與m303不存在於台灣族群，而是有其他因子可以防止台灣人不被HIV-1感染。
除了CCR5突變基因的偵測之外，我們想要瞭解是否有其他的基因或細胞因子可以保護中國人不被HIV-1感染。為了達到這個目的，我們將注意力放在多次接觸到HIV-1卻不被病毒感染的人之族群，我們首先鑑定出他們HLA class I及class II的基因型，當中如A2、DQ7出現頻率很高，在台灣族群很有可能是與不易被HIV-1感染的基因有關。由於在西方國家的一些種族中已經證明血清高濃度的b-chemokines與IL-16能防止HIV-1的感染及抑制HIV-1的繁殖，於是我們設計了一連串的實驗來測定這些不被HIV-1感染者血清內及周邊血液淋巴細胞(peripheral blood lymphocytes 簡稱PBL)分泌出b-chemokines與IL-16的濃度。首先，我們將他們全血中的PBL取出並施以細胞培養後，再以PHA刺激或單純培養。然後收集細胞培養液去做酵素免疫顯色法(ELISA)分析，測定PBL分泌的b-chemokines與IL-16之濃度。由9位不被HIV-1感染者以及6位正常人的血清及PBL分泌於培養液之ELISA分析結果得知，不被HIV-1感染者血清中MIP-1a、 MIP-1b與 RANTES的濃度都比未暴露於HIV-1的正常人明顯來得高，同時不被HIV-1感染者的PBL在細胞培養後經PHA刺激過的培養液中MIP-1a的濃度也明顯較高。另外我們分析了經細胞培養後PBL之b-chemokines與IL-16的mRNA表現量，然而與蛋白質的量做比較並沒有相關性。
以上的結果獲得的結論是：不被HIV-1感染者體內的b-chemokines比未暴露於此病毒的正常人明顯有較高的表現量。由於目前我們並沒有發現其他明顯可以保護台灣人不被HIV-1感染的基因，因此b-chemokines很可能是防止台灣族群不被HIV-1感染最重要的因子。

[ 英文摘要 ]

HIV-1 is a human retrovirus. It enters the target cells by fusion with the plasma membrane through interaction of viral envelope proteins and cellular receptors. The primary receptor for HIV-1 entry is CD4 and the coreceptor utilization is depended on the tropism of HIV-1 strains. HIV-1 isolates that infect macrophages and primary T cells (called M-tropic or R5 strains) use the b-chemokine receptor CCR5 as the coreceptor, whereas strains that infect primary T cells and transformed T cell lines (called T-tropic or X4 strains) use the a-chemokine receptor CXCR4. The previous studies have showed that HIV-1 coreceptors and their nature ligands play the critical roles for the HIV-1 pathogenesis. For example, HIV-1 cannot infect Caucasians who are homozygous for the mutated HIV-1 coreceptor ccr5 gene (D32, for 32-bp deletion in the ccr5 coding region) or have the high levels of serum suppressive factors, such as b-chemokines (MIP-1a, MIP-1b and RANTES) and interleukin (IL)-16. Although D32 and the elevated levels of b-chemokines and IL-16 have been demonstrated to block HIV-1 infection, the allele and the cellular factors have never been systematically characterized in Chinese, the largest ethnic population in the world. Therefore, I first surveyed the D32 allele in 369 individuals in Taiwan, which included HIV-1-uninfected, high-risk, infected, and normal populations, but failed to find any D32 allele. I further characterized another reported ccr5 mutant allele (m303) in Taiwanese, which also play a protective role against the transmission of the M-tropic HIV-1 strains. Like the D32 allele, I did not find any m303 allele in the studied group (n=316). The results demonstrate that CCR5 D32 and m303 do not exist in Taiwanese and there are other factors to block HIV-1 infection.
Besides the ccr5 mutated allele determinations, I investigated other genes or cellular factors that may protect Taiwanese from HIV-1 infection. To do this, I focused my attention on the immunity of a cohort of nine HIV-1-uninfected individuals who have multiply-exposed to the virus. I first investigated their HLA class I and class II types and revealed that some alleles exhibited a higher frequency which may have a correlation to HIV-1 infection, such as the class I A2 allele and the class II DQ7 allele. Since high serum levels of b-chemokines and IL-16 have been demonstrated to protect several western populations from HIV-1 infection and inhibit viral replication, respectively. I designed an experimental protocol to determine amounts of b-chemokines and IL-16 secreted by peripheral blood lymphocytes (PBL) and in serum from these individuals. I first isolated and cultured their PBL in vitro, with or without stimulation. The supernatants were then collected and analyzed for the protein concentrations as determined by enzyme-linked immunosorbent assay (ELISA). From ELISA analysis of these nine HIV-1-uninfected individuals and six HIV-1-unexposed control individuals, I found that the serum concentrations of MIP-1a, MIP-1b and RANTES for HIV-1-uninfected individuals were significantly higher than the unexposed controls. A significant higher amount of MIP-1a for uninfected cohort was also found in stimulated culture supernatants. In parallel, I analyzed the mRNA expression for b-chemokines and IL-16 in the culture PBL. However, no correlation was found.
In conclusion, higher levels of serum b-chemokines were detected in the HIV-1-uninfected cohort as compared to unexposed control. Since no other protective alleles have been found so far in Taiwanese, therefore, the b-chemokines may play an important role in defending Taiwanese against HIV-1 infection.