本文選題：膿毒癥 + 盲腸結(jié)扎穿孔��； 參考：《第二軍醫(yī)大學(xué)》2013年博士論文

【摘要】：【研究目的】 膿毒癥是感染引起的全身炎癥反應(yīng)綜合征，死亡率一直居高不下。免疫抑制是膿毒癥后期常見(jiàn)的免疫異常狀態(tài)。巨噬細(xì)胞功能障礙是這種免疫抑制的一個(gè)重要原因，但是其具體發(fā)生機(jī)制目前尚不明了。 程序性死亡分子1（programmed cell death1, PD-1）是近年來(lái)發(fā)現(xiàn)的共抑制受體，參與外周組織免疫耐受和慢性病毒感染。PD-1基因敲除可以顯著提高膿毒癥小鼠的生存率，提示PD-1在膿毒癥免疫抑制中發(fā)揮著重要的作用。本課題擬首先檢測(cè)PD-1在肝臟枯否細(xì)胞的表達(dá)，進(jìn)而應(yīng)用PD-1基因敲除小鼠，觀察PD-1在膿毒癥肝臟枯否細(xì)胞功能障礙的作用，并初步探索PD-1在枯否細(xì)胞發(fā)揮作用的分子機(jī)制及PD-1參與膿毒癥多器官功能障礙的分子機(jī)制。 【研究方法】 1、復(fù)制盲腸結(jié)扎穿孔（cecal ligation and puncture, CLP）致膿毒癥小鼠模型。將16只野生型C57BL/6小鼠隨機(jī)分為假手術(shù)組（Sham, n=8）和CLP組（CLP, n=8）。術(shù)后24h時(shí)取肝臟，分離肝臟非實(shí)質(zhì)細(xì)胞，應(yīng)用流式細(xì)胞儀檢測(cè)枯否細(xì)胞（F4/80+）表面PD-1的表達(dá)水平。 2、將10只野生型C57BL/6小鼠及10只PD-1基因敲除小鼠隨機(jī)分為野生型假手術(shù)組（WT Sham, n=4），PD-1基因敲除假手術(shù)組（PD-1-/-Sham, n=4），野生型CLP組（WT CLP, n=6）和PD-1基因敲除CLP組（PD-1-/-CLP, n=6）。術(shù)后24h時(shí)，分離肝臟非實(shí)質(zhì)細(xì)胞，應(yīng)用流式細(xì)胞儀檢測(cè)枯否細(xì)胞表面MHCΠ、CD69、CD80和CD86的表達(dá)水平。 3、將8只野生型C57BL/6小鼠及8只PD-1基因敲除小鼠隨機(jī)分為野生型假手術(shù)組（WT Sham, n=4），PD-1基因敲除假手術(shù)組（PD-1-/-Sham, n=4），野生型CLP組（WT CLP, n=4）和PD-1基因敲除CLP組（PD-1-/-CLP, n=4）。術(shù)后24h時(shí)，分離肝臟非實(shí)質(zhì)細(xì)胞，貼壁法純化枯否細(xì)胞。加入分子探針pHrodoTME. coli BioParticlesConjugate共培養(yǎng)1h，應(yīng)用流式細(xì)胞儀檢測(cè)枯否細(xì)胞吞噬功能。 4、將10只野生型C57BL/6小鼠及10只PD-1基因敲除小鼠隨機(jī)分為野生型假手術(shù)組（WT Sham, n=4），PD-1基因敲除假手術(shù)組（PD-1-/-Sham, n=4），野生型CLP組（WT CLP, n=6）和PD-1基因敲除CLP組（PD-1-/-CLP, n=6）。術(shù)后24h時(shí)，分離肝臟非實(shí)質(zhì)細(xì)胞，貼壁法純化枯否細(xì)胞。LPS（1μg/ml）刺激或不刺激24h后收集培養(yǎng)上清及細(xì)胞，ELISA法檢測(cè)細(xì)胞因子TNF-α、IL-1β、IL-6、IL-10、IL-12p40和MCP-1水平。 5、將10只野生型C57BL/6小鼠及10只PD-1基因敲除小鼠隨機(jī)分為野生型假手術(shù)組（WT Sham, n=4），PD-1基因敲除假手術(shù)組（PD-1-/-Sham, n=4），野生型CLP組（WT CLP, n=6）和PD-1基因敲除CLP組（PD-1-/-CLP, n=6）。術(shù)后24h時(shí)，分離肝臟非實(shí)質(zhì)細(xì)胞，貼壁法純化枯否細(xì)胞。LPS（1μg/ml）刺激24h后收集細(xì)胞，Westernblot法檢測(cè)cleaved caspase-3的水平。 6、將10只野生型C57BL/6小鼠及10只PD-1基因敲除小鼠隨機(jī)分為野生型假手術(shù)組（WT Sham, n=4），PD-1基因敲除假手術(shù)組（PD-1-/-Sham, n=4），野生型CLP組（WT CLP, n=6）和PD-1基因敲除CLP組（PD-1-/-CLP, n=6）。術(shù)后24h時(shí)，分離肝臟非實(shí)質(zhì)細(xì)胞，貼壁法純化枯否細(xì)胞。LPS（1μg/ml）刺激24h后收集細(xì)胞，TUNEL法檢測(cè)凋亡情況。 7、將10只野生型C57BL/6小鼠及10只PD-1基因敲除小鼠隨機(jī)分為野生型假手術(shù)組（WT Sham, n=4），PD-1基因敲除假手術(shù)組（PD-1-/-Sham, n=4），野生型CLP組（WT CLP, n=6）和PD-1基因敲除CLP組（PD-1-/-CLP, n=6）。術(shù)后24h時(shí)，分離肝臟非實(shí)質(zhì)細(xì)胞，貼壁法純化枯否細(xì)胞。LPS（1μg/ml）刺激10min后收集細(xì)胞，Western blot法檢測(cè)磷酸化Akt及總Akt水平。 8、將10只野生型C57BL/6小鼠及10只PD-1基因敲除小鼠隨機(jī)分為野生型假手術(shù)組（WT Sham, n=4），PD-1基因敲除假手術(shù)組（PD-1-/-Sham, n=4），野生型CLP組（WT CLP, n=6）和PD-1基因敲除CLP組（PD-1-/-CLP, n=6）。術(shù)后24h時(shí)，分離肝臟非實(shí)質(zhì)細(xì)胞，貼壁法純化枯否細(xì)胞。LPS（1μg/ml）刺激10min后收集細(xì)胞，Western blot法檢測(cè)磷酸化p38及總p38水平。 9、將10只野生型C57BL/6小鼠及10只PD-1基因敲除小鼠隨機(jī)分為野生型假手術(shù)組（WT Sham, n=4），PD-1基因敲除假手術(shù)組（PD-1-/-Sham, n=4），野生型CLP組（WT CLP, n=6）和PD-1基因敲除CLP組（PD-1-/-CLP, n=6）。術(shù)后24h時(shí)，取心臟、肝臟和腎臟。組織勻漿，Western blot法檢測(cè)磷酸化Akt、總Akt、磷酸化STAT3和總STAT3水平。 【結(jié)果】 1、術(shù)后24h時(shí)，膿毒癥小鼠肝臟枯否細(xì)胞表面PD-1表達(dá)顯著高于假手術(shù)組（P0.001）. 2、術(shù)后24h時(shí)，野生型膿毒癥小鼠肝臟枯否細(xì)胞表面MHCΠ、CD86表達(dá)均顯著低于野生型假手術(shù)組（P 0.001），CD80表達(dá)顯著高于野生型假手術(shù)組（P 0.001）。PD-1基因敲除可部分但顯著恢復(fù)枯否細(xì)胞表面MHCΠ、CD80和CD86表達(dá)（P 0.001）。各組CD69表達(dá)無(wú)顯著改變。 3、術(shù)后24h時(shí)，野生型膿毒癥小鼠肝臟枯否細(xì)胞吞噬功能顯著下降（P 0.01），PD-1基因敲除膿毒癥小鼠可顯著增強(qiáng)枯否細(xì)胞功能（P 0.001）。 4、術(shù)后24h分離肝臟枯否細(xì)胞，體外LPS繼續(xù)刺激或不刺激24h時(shí)，野生型膿毒癥小鼠肝臟枯否細(xì)胞分泌TNF-α、IL-1β、IL-6、IL-10、IL-12p40和MCP-1能力顯著下降（P 0.05）。PD-1基因敲除可部分但顯著恢復(fù)枯否細(xì)胞分泌上述細(xì)胞因子能力。 5、術(shù)后24h分離肝臟枯否細(xì)胞，體外LPS繼續(xù)刺激24h時(shí)，野生型膿毒癥小鼠肝臟枯否細(xì)胞cleaved caspase-3水平顯著升高（P 0.001），PD-1基因敲除可顯著減少枯否細(xì)胞cleaved caspase-3水平（P 0.001）。 6、術(shù)后24h分離肝臟枯否細(xì)胞，體外LPS繼續(xù)刺激24h時(shí)，野生型膿毒癥小鼠肝臟枯否細(xì)胞TUNEL染色陽(yáng)性比例顯著升高，PD-1基因敲除可顯著減少枯否細(xì)胞TUNEL染色陽(yáng)性比例。 7、術(shù)后24h分離肝臟枯否細(xì)胞，體外LPS繼續(xù)刺激10min時(shí)，，野生型膿毒癥小鼠肝臟枯否細(xì)胞磷酸化Akt/總Akt比例顯著降低（P 0.001），PD-1基因敲除可顯著增加枯否細(xì)胞磷酸化Akt/總Akt比例（P 0.001）。 8、術(shù)后24h分離肝臟枯否細(xì)胞，體外LPS繼續(xù)刺激10min時(shí)，野生型膿毒癥小鼠肝臟枯否細(xì)胞磷酸化p38/總p38比例顯著升高（P 0.001），PD-1基因敲除可顯著減少枯否細(xì)胞磷酸化p38/總p38比例（P 0.001）。 9、術(shù)后24h時(shí)，野生型膿毒癥小鼠心臟、肝臟和腎臟磷酸化Akt/總Akt比例顯著降低，PD-1基因敲除可顯著增加肝臟和腎臟磷酸化Akt/總Akt比例，但心臟磷酸化Akt/總Akt比例無(wú)顯著變化。野生型膿毒癥小鼠心臟、肝臟和腎臟磷酸化STAT3/總STAT3比例顯著升高，PD-1基因敲除可顯著減少心臟和腎臟磷酸化STAT3/總STAT3比例，但是肝臟STAT3/總STAT3比例無(wú)顯著變化。 【結(jié)論】 PD-1參與了膿毒癥導(dǎo)致的肝臟枯否細(xì)胞功能障礙，可能通過(guò)影響枯否細(xì)胞Akt磷酸化和p38磷酸化發(fā)揮作用，還可能通過(guò)影響Akt磷酸化和STAT3磷酸化參與膿毒癥導(dǎo)致的多器官功能障礙。
[Abstract]:[purpose]
Sepsis is a systemic inflammatory reaction caused by infection syndrome, mortality is high. The immunosuppressive state is immune abnormalities common sepsis later. Macrophage dysfunction is an important reason for this immune suppression, but the specific mechanism is unclear.
Programmed cell death 1 (programmed cell, death1, PD-1) is a co inhibitory receptor found in recent years, in the knockout can significantly improve the survival rate of sepsis mice.PD-1 gene in peripheral tissues and immune tolerance in chronic viral infection, suggesting that PD-1 in sepsis immunosuppression plays an important role in this paper. The first detection of PD-1 in Kupffer cells and expression of PD-1 gene knockout mice, to observe the effect of PD-1 on sepsis Kupffer cell dysfunction, and to explore the molecular mechanism and molecular mechanism of PD-1 play the role of PD-1 involved in sepsis and multiple organ dysfunction in Kupffer cells.
[research methods]
1, copy the cecal ligation and perforation (cecal ligation and puncture, CLP) induced sepsis mice model. 16 male wild-type C57BL/6 mice were randomly divided into sham operation group (Sham, n=8) and CLP group (CLP, n=8). After 24h, the liver and isolated liver non parenchymal cells, detect Kupffer by flow cytometry (F4/80+) expression level of PD-1 on the surface.
2, 10 wild type C57BL/6 mice and 10 PD-1 knockout mice were randomly divided into sham operation group (wild type WT Sham, n=4), PD-1 knockout sham operation group (PD-1-/-Sham, n=4), wild type CLP group (WT CLP, n=6) and PD-1 knockout (PD-1-/-CLP CLP group n=6, 24h). After the operation, the separation of liver nonparenchymal cells, Kupffer cell surface MHC PI assay, flow cytometry was used to CD69, the expression level of CD80 and CD86.
3, 8 wild type C57BL/6 mice and 8 PD-1 knockout mice were randomly divided into sham operation group (wild type WT Sham, n=4), PD-1 knockout sham operation group (PD-1-/-Sham, n=4), wild type CLP group (WT CLP, n=4) and PD-1 knockout (PD-1-/-CLP CLP group n=4, 24h). After the operation, the separation of liver nonparenchymal cells, adhesion purified Kupffer cells. The molecular probe pHrodoTME. coli BioParticlesConjugate joined the co culture 1H, flow cytometer was used to detect the phagocytosis of Kupffer cells.
4, 10 wild type C57BL/6 mice and 10 PD-1 knockout mice were randomly divided into sham operation group (wild type WT Sham, n=4), PD-1 knockout sham operation group (PD-1-/-Sham, n=4), wild type CLP group (WT CLP, n=6) and PD-1 knockout (PD-1-/-CLP CLP group n=6, 24h). After the operation, the separation and purification of liver non parenchymal cells and Kupffer cells adherent.LPS (1 g/ml) stimulated or not after 24h stimulation and cell culture supernatants were collected, detection of cytokines TNF-, ELISA IL-1 IL-6, IL-10, beta, IL-12p40 and MCP-1 levels.
5, 10 wild type C57BL/6 mice and 10 PD-1 knockout mice were randomly divided into sham operation group (wild type WT Sham, n=4), PD-1 knockout sham operation group (PD-1-/-Sham, n=4), wild type CLP group (WT CLP, n=6) and PD-1 knockout (PD-1-/-CLP CLP group n=6, 24h). After the operation, the separation and purification of liver non parenchymal cells and Kupffer cells adherent.LPS (1 g/ml) stimulation of 24h cells were collected after detection of cleaved caspase-3 by Westernblot.
6, 10 wild type C57BL/6 mice and 10 PD-1 knockout mice were randomly divided into sham operation group (wild type WT Sham, n=4), PD-1 knockout sham operation group (PD-1-/-Sham, n=4), wild type CLP group (WT CLP, n=6) and PD-1 knockout (PD-1-/-CLP CLP group n=6, 24h). After the operation, the separation and purification of liver non parenchymal cells and Kupffer cells adherent.LPS (1 g/ml) after stimulation of 24h cells were collected to detect the apoptosis of TUNEL.
7, 10 wild type C57BL/6 mice and 10 PD-1 knockout mice were randomly divided into sham operation group (wild type WT Sham, n=4), PD-1 knockout sham operation group (PD-1-/-Sham, n=4), wild type CLP group (WT CLP, n=6) and PD-1 knockout (PD-1-/-CLP CLP group n=6, 24h). After the operation, the separation and purification of liver non parenchymal cells and Kupffer cells adherent.LPS (1 g/ml) stimulation of 10min cells were collected after Western blot detection of phosphorylated Akt and total Akt levels.
8, 10 wild type C57BL/6 mice and 10 PD-1 knockout mice were randomly divided into sham operation group (wild type WT Sham, n=4), PD-1 knockout sham operation group (PD-1-/-Sham, n=4), wild type CLP group (WT CLP, n=6) and PD-1 knockout (PD-1-/-CLP CLP group n=6, 24h). After the operation, the separation and purification of liver non parenchymal cells and Kupffer cells adherent.LPS (1 g/ml) stimulation of 10min cells were collected after Western blot detection of phosphorylated p38 and total p38 levels.
9, 10 wild type C57BL/6 mice and 10 PD-1 knockout mice were randomly divided into sham operation group (wild type WT Sham, n=4), PD-1 knockout sham operation group (PD-1-/-Sham, n=4), wild type CLP group (WT CLP, n=6) and PD-1 knockout (PD-1-/-CLP CLP group n=6, 24h). After the operation, take heart, liver and kidney homogenates. Detection of phosphorylated Akt, Western, blot total Akt, phosphorylated STAT3 and total STAT3 levels.
[results]
1, the expression of PD-1 on the surface of liver Kupffer cells in sepsis mice was significantly higher than that of the sham operation group (P0.001) at 24h after operation.
2, after 24h, the wild type of sepsis mice Kupffer cell surface MHC PI, the expression of CD86 was significantly lower than that of the wild type in sham operation group (P 0.001), the expression of CD80 was significantly higher than that of the wild type in sham operation group (P 0.001).PD-1 gene knockout partially but significantly restored Kupffer cell surface MHC Pi the expression of CD80 and CD86 (P, 0.001). The expression of CD69 in each group had no significant change.
3, after 24h, the phagocytic function of Kupffer cells in wild type sepsis mice was significantly decreased (P 0.01). PD-1 knockout mice could significantly enhance the function of Kupffer cells (P 0.001).
4, 24h after separation of Kupffer cells in vitro, LPS continues to stimulate or not to stimulate the 24h, wild type septic mice Kupffer cells secrete TNF- alpha, IL-1 beta, IL-6, IL-10, IL-12p40 and MCP-1 decreased significantly (P 0.05).PD-1 gene knockout partially but significantly restored the secretion of dry cytokines in Kupffer cells.
5, after 24h, the hepatic Kupffer cells were separated from the liver. When LPS continued to stimulate 24h in vitro, the cleaved caspase-3 level of Kupffer cells in wild type sepsis mice increased significantly (P 0.001). PD-1 knockout significantly reduced the cleaved caspase-3 level of Kupffer cells (P 0.001).
6, after 24h, the hepatic Kupffer cells were separated from the liver. When LPS continued to stimulate 24h in vitro, the positive rate of TUNEL staining in Kupffer cells of wild type sepsis mice increased significantly. PD-1 knockout significantly reduced the positive proportion of TUNEL staining in Kupffer cells.
7, after 24h, the hepatic Kupffer cells were separated from the liver. When LPS continued to stimulate 10min in vitro, the proportion of total Akt/ Akt in Kupffer cells in the wild type sepsis mice decreased significantly (P 0.001). PD-1 knockout significantly increased the percentage of Akt/ Akt in Kupffer cells (P 0.001).
8, after 24h, the liver Kupffer cells were separated from the liver. When LPS continued to stimulate 10min in vitro, the proportion of p38/ p38 in Kupffer cells of wild type sepsis mice increased significantly (P 0.001). PD-1 knockout significantly reduced the percentage of p38/ p38 in Kupffer cells (P 0.001).
9, after 24h, the wild type of sepsis mice heart, liver and kidney of phosphorylated Akt/ total Akt ratio was significantly decreased, PD-1 knockout significantly increased the phosphorylation of Akt/ in liver and kidney total Akt ratio, but the heart of the phosphorylation of Akt/ ratio of total Akt. No significant changes in wild type sepsis mouse heart the liver and kidney of phosphorylated STAT3/ and total STAT3 ratio was significantly increased, PD-1 knockout significantly reduced the phosphorylation of STAT3/ in heart and kidney total STAT3 ratio, but the liver STAT3/ ratio of total STAT3 showed no significant changes.
[Conclusion]
PD-1 participates in sepsis induced Kupffer cell dysfunction. It may play a role in the Akt phosphorylation and p38 phosphorylation of Kupffer cells. It may also play a role in multiple organ dysfunction caused by sepsis by affecting Akt phosphorylation and STAT3 phosphorylation.

【學(xué)位授予單位】：第二軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2013
【分類(lèi)號(hào)】：R459.7

【共引文獻(xiàn)】

相關(guān)期刊論文 前10條

1 官杰;吳艷敏;王慧;錢(qián)麗麗;李殿俊;;殼寡糖協(xié)同雙歧桿菌對(duì)荷瘤鼠免疫功能的影響[J];癌變.畸變.突變;2007年06期

2 何江帥;盧強(qiáng);李偉;趙曉;馮祥汝;陳義龍;;鯉魚(yú)白細(xì)胞介素-1β全長(zhǎng)cDNA的克隆·鑒定及其差異表達(dá)分析[J];安徽農(nóng)業(yè)科學(xué);2011年12期

3 何雁;王啟之;;Toll樣受體9與潰瘍性結(jié)腸炎[J];安徽醫(yī)藥;2009年06期

4 何雁;王志紅;楊善峰;石振旺;李祥;代多珍;;TLR9和NF-κBp65在大鼠潰瘍性結(jié)腸炎模型結(jié)腸組織中的表達(dá)[J];安徽醫(yī)藥;2012年07期

5 盛榮華;陳靠山;;多糖對(duì)腸道黏膜免疫的影響及其抗腫瘤活性研究進(jìn)展[J];現(xiàn)代農(nóng)業(yè)科技;2011年01期

6 毛詩(shī)海;程訓(xùn)民;楊松;葛玲;崔杰西;;尿毒癥維持性血液透析患者超敏C反應(yīng)蛋白檢測(cè)臨床應(yīng)用[J];蚌埠醫(yī)學(xué)院學(xué)報(bào);2012年03期

7 時(shí)小紅;徐珊;;萎縮性胃炎脾氣虛證免疫小分子蛋白組表達(dá)的研究[J];浙江中醫(yī)藥大學(xué)學(xué)報(bào);2012年05期

8 宓偉;;天麻增強(qiáng)小鼠免疫功能的實(shí)驗(yàn)研究[J];濱州醫(yī)學(xué)院學(xué)報(bào);2010年04期

9 高曉寧;姚瑜;高翔;藺志清;毛穎;;膠質(zhì)瘤免疫逃逸機(jī)制與放療的關(guān)系研究進(jìn)展[J];中國(guó)神經(jīng)腫瘤雜志;2011年01期

10 賈麗;高建;;藥物誘導(dǎo)自身免疫性肝炎發(fā)病機(jī)制的研究進(jìn)展[J];重慶醫(yī)學(xué);2008年08期

相關(guān)會(huì)議論文 前10條

1 羅政;魏霞;熊章華;;中藥在直腸癌術(shù)后放療中防護(hù)作用的臨床研究[A];江西省首屆中西醫(yī)結(jié)合腫瘤學(xué)術(shù)研討會(huì)論文集[C];2010年

2 彭界;;中老年呼吸道疾病患者M(jìn)P-IgM檢測(cè)分析[A];第二十五屆航天醫(yī)學(xué)年會(huì)暨第八屆航天護(hù)理年會(huì)論文匯編[C];2009年

3 張景科;程杰;席曉霞;田永剛;;SPF級(jí)Wistar大鼠生產(chǎn)管理技術(shù)要點(diǎn)[A];第九屆中國(guó)實(shí)驗(yàn)動(dòng)物科學(xué)年會(huì)（2010新疆）論文集[C];2010年

4 潘明沃;陳志強(qiáng);古熾明;王樹(shù)聲;向松濤;呂立國(guó);代睿欣;;前列腺癌浸潤(rùn)性樹(shù)突狀細(xì)胞CD1a、CD83的表達(dá)及與臨床指標(biāo)的相關(guān)性分析[A];第七次中國(guó)中西醫(yī)結(jié)合泌尿外科學(xué)術(shù)年會(huì)暨第二次廣東省中西醫(yī)結(jié)合泌尿外科學(xué)術(shù)年會(huì)論文集[C];2009年

5 潘明沃;陳志強(qiáng);古熾明;呂立國(guó);代睿欣;;“扶正抑瘤法”對(duì)前列腺癌患者原發(fā)灶樹(shù)突狀細(xì)胞CD1a、CD83表達(dá)的影響[A];第七次中國(guó)中西醫(yī)結(jié)合泌尿外科學(xué)術(shù)年會(huì)暨第二次廣東省中西醫(yī)結(jié)合泌尿外科學(xué)術(shù)年會(huì)論文集[C];2009年

6 潘明沃;陳志強(qiáng);古熾明;王樹(shù)聲;向松濤;呂立國(guó);代睿欣;;前列腺癌浸潤(rùn)性樹(shù)突狀細(xì)胞CD1a CD83的表達(dá)及與臨床指標(biāo)的相關(guān)性分析[A];第七次中國(guó)中西醫(yī)結(jié)合泌尿外科學(xué)術(shù)年會(huì)暨第二次廣東省中西醫(yī)結(jié)合泌尿外科學(xué)術(shù)年會(huì)論文集[C];2009年

7 周立華;唐英;黃朝陽(yáng);郭會(huì)軍;;從中醫(yī)整體論角度探討艾滋病非CD4+T細(xì)胞的免疫補(bǔ)償機(jī)制[A];中華中醫(yī)藥學(xué)會(huì)防治艾滋病學(xué)術(shù)研討會(huì)暨2006年年會(huì)論文集[C];2006年

8 蘇愛(ài)華;叢U

本文編號(hào)：1762758