人Tim-3啟動(dòng)子的克隆及其逆轉(zhuǎn)錄病毒表達(dá)載體的構(gòu)建
發(fā)布時(shí)間：2018-09-01 07:11
【摘要】： Tim-3(T-cell immunoglobulin-and mucin-domain-containing molecule-3,T細(xì)胞免疫球蛋白粘蛋白分子-3)是新近發(fā)現(xiàn)的免疫調(diào)節(jié)分子,特異表達(dá)于分化的Th1細(xì)胞,參與Th1負(fù)調(diào)控和免疫耐受形成,在多種免疫性疾病發(fā)生中發(fā)揮重要作用,阻斷其表達(dá)可緩解Th2應(yīng)答引起的過敏性疾病,是多種免疫性疾病的潛在治療靶標(biāo),引起國內(nèi)外學(xué)者廣泛關(guān)注。但Tim-3研究尚存在諸多問題未能解決,尤其是其表達(dá)調(diào)控機(jī)制研究尚屬空白。本研究分三部分內(nèi)容:初步研究正常人PBMC Tim-3的表達(dá)模式;克隆具有特異啟動(dòng)子活性的基因片段,構(gòu)建人Tim-3啟動(dòng)子報(bào)告質(zhì)粒;構(gòu)建人Tim-3逆轉(zhuǎn)錄病毒表達(dá)載體,為進(jìn)一步研究Tim-3表達(dá)調(diào)控機(jī)制及探索利用Tim-3進(jìn)行疾病治療的可行性奠定基礎(chǔ)。 第一部分正常人PBMC Tim-3表達(dá)模式的初步研究 研究目的 檢測經(jīng)絲裂原刺激的人外周血淋巴細(xì)胞Tim-3的表達(dá)情況,為進(jìn)一步研究Tim-3表達(dá)調(diào)控機(jī)制提供實(shí)驗(yàn)?zāi)Ｐ汀?研究方法 分離人PBMC,貼壁法去除單核細(xì)胞后,分別以PHA、LPS刺激懸浮的淋巴細(xì)胞,24h后抽提細(xì)胞總RNA,RT-PCR檢測Tim-3的表達(dá)。未刺激淋巴細(xì)胞作對(duì)照。 研究結(jié)果 RT-PCR檢測顯示PHA刺激的人外周血淋巴細(xì)胞Tim-3表達(dá)上調(diào),而LPS刺激的不表達(dá)Tim-3,提示處于活化、非極化的T細(xì)胞就開始表達(dá)該分子。 結(jié)論及意義 本部分建立了可供作為研究Tim-3表達(dá)調(diào)控的細(xì)胞模型,為進(jìn)一步深入研究Tim-3表達(dá)調(diào)控機(jī)制提供實(shí)驗(yàn)基礎(chǔ)。 第二部分人Tim-3啟動(dòng)子的克隆及其活性的初步研究 研究目的 構(gòu)建人Tim-3啟動(dòng)子螢火蟲熒光素酶報(bào)告質(zhì)粒并分析其活性,為進(jìn)一步研究Tim-3特異表達(dá)于Th1細(xì)胞的分子機(jī)制奠定基礎(chǔ)。 研究方法 1人Tim-3啟動(dòng)子區(qū)的預(yù)測 利用在線MatInspector軟件對(duì)人Tim-3基因5′端-2500～+202段進(jìn)行啟動(dòng)子區(qū)預(yù)測。 2 Tim-3P1與Tim-3P2 PCR擴(kuò)增 選定長2325bp的-2083～+242段,并依據(jù)該段單一SacⅠ酶切位點(diǎn),分Tim-3P1(-2083～-838)和Tim-3P2(-948～+242)兩部分克隆。設(shè)計(jì)針對(duì)Tim-3P1和Tim-3P2的特異性引物,以人基因組DNA為模板進(jìn)行PCR擴(kuò)增。 3含不同長度人Tim-3基因5′端片段報(bào)告質(zhì)粒的構(gòu)建及鑒定 Tim-3P1 PCR產(chǎn)物,T-A克隆入pGEM-T,測序正確后,亞克隆入pGL3-Basic相應(yīng)酶切位點(diǎn)構(gòu)建pGL3-Tim-3P1。Tim-3P2 PCR產(chǎn)物,經(jīng)SacⅠ、XhoⅠ雙酶切、回收后,插入pGL3-Basic多克隆位點(diǎn)(MCS)構(gòu)建pGL3-Tim-3P2。亦經(jīng)測序鑒定。為增強(qiáng)所構(gòu)建報(bào)告質(zhì)粒的啟動(dòng)子活性,設(shè)計(jì)在上述陽性pGL3-Tim-3P2中引入SV40增強(qiáng)子序列構(gòu)建Enhancer-Tim-3P2,經(jīng)雙酶切鑒定。 4 RT-PCR檢測細(xì)胞系內(nèi)源性Tim-3的表達(dá) 收集對(duì)數(shù)生長期RAW264.7、B16及COS-75×10~5,Trizol法抽提細(xì)胞總RNA并行RT-PCR檢測。 5報(bào)告質(zhì)粒的轉(zhuǎn)染 將上述構(gòu)建的報(bào)告質(zhì)粒及pGL3-Basic(陰性對(duì)照)分別與pRL-TK(內(nèi)參照)瞬時(shí)共轉(zhuǎn)染RAW264.7、B16和COS-7(詳見附圖表第二部分),48h收集細(xì)胞,對(duì)細(xì)胞粗提液進(jìn)行雙熒光素酶分析。 6雙熒光素酶分析 按雙熒光素酶報(bào)告基因檢測試劑盒說明書將細(xì)胞裂解后與底物混合,用熒光計(jì)數(shù)儀檢測所構(gòu)建報(bào)告質(zhì)粒中螢火蟲熒光素酶活性M1與pRL-TK中海腎熒光素酶活性M2,M1/M2的比值即為目的片段的相對(duì)活性。每種質(zhì)粒設(shè)2復(fù)孔,同一轉(zhuǎn)染實(shí)驗(yàn)至少重復(fù)3次。 7 Tim-3P1與Tim-3P2轉(zhuǎn)錄因子結(jié)合位點(diǎn)(TFBSs)分析 同樣利用在線MatInspector軟件分析Tim-3P1與Tim-3P2中可能存在的TFBSs。 研究結(jié)果 1人Tim-3啟動(dòng)子區(qū)的預(yù)測 分析顯示-2500～+202段存在多個(gè)TATA box、CAAT box及多個(gè)重要TFBSs,可能具有啟動(dòng)子活性。 2成功構(gòu)建人Tim-3啟動(dòng)子螢火蟲熒光素酶報(bào)告質(zhì)粒 測序鑒定證實(shí)pGL3-Tim-3P1、pGL3-Tim-3P2構(gòu)建成功;酶切鑒定證實(shí)Enhancer-Tim-3P2構(gòu)建成功。 3不同細(xì)胞系內(nèi)源性Tim-3的表達(dá) RT-PCR檢測顯示RAW264.7和B16表達(dá)內(nèi)源性Tim-3,而COS-7不表達(dá)。 4報(bào)告質(zhì)粒啟動(dòng)子活性的驗(yàn)證 雙熒光素酶分析證實(shí)pGL3-Tim-3P1與pGL3-Tim-3P2均具有較弱的啟動(dòng)子活性,其中,pGL3-Tim-3P2相對(duì)活性約是pGL3-Basic空載體的2倍,且啟動(dòng)子活性具有細(xì)胞特異性;引入增強(qiáng)子的Enhancer-Tim-3P2啟動(dòng)子活性明顯升高。 5 TFBSs分析 結(jié)果顯示Tim-3P1含有下列TFBSs:①1個(gè)c-Myb、Ik-1、YY1(Yin and Yang 1,陰陽1)、GATA-3(GATA-binding factor-3)和CHOP;②2個(gè)FOXK2、HNF-3(hepatocyte nuclear factor-3,肝細(xì)胞核因子-3);③6個(gè)TATA box;Tim-3P2含有下列TFBSs:①1個(gè)RFX1、p53、GATA-3、NFAT(Nuclear factor of activated T cells,活化T細(xì)胞核因子)和CDE/CHR(cell cycle-dependentelement,細(xì)胞周期依賴性元件;cell cycle genes homology region,細(xì)胞周期基因同源區(qū));②2個(gè)MZF1(Myeloid zinc finger protein-1);③3個(gè)YY1;④5個(gè)TATA box。 結(jié)論及意義 本部分成功構(gòu)建了含人Tim-3啟動(dòng)子的螢火蟲熒光素酶報(bào)告質(zhì)粒和帶有增強(qiáng)子序列的報(bào)告質(zhì)粒,為進(jìn)一步研究Tim-3表達(dá)調(diào)控機(jī)制奠定了基礎(chǔ)。鑒于Tim-3在免疫應(yīng)答中的重要作用,深入研究其表達(dá)調(diào)控將具有重要的理論意義和良好的應(yīng)用前景。 第三部分人Tim-3逆轉(zhuǎn)錄病毒表達(dá)載體的構(gòu)建 研究目的 構(gòu)建人Tim-3逆轉(zhuǎn)錄病毒表達(dá)載體,為進(jìn)一步研究Tim-3表達(dá)調(diào)控機(jī)制、探索利用Tim-3治療疾病的可行性奠定基礎(chǔ)。 研究方法 設(shè)計(jì)針對(duì)人Tim-3 mRNA的特異性引物,以hTim-3-pGEM-T為模板進(jìn)行PCR擴(kuò)增。產(chǎn)物克隆入逆轉(zhuǎn)錄病毒表達(dá)載體pLNCX2的HindⅢ、NotⅠ位點(diǎn),構(gòu)建pLNCX2-Tim-3。經(jīng)PCR、酶切及測序鑒定正確后,轉(zhuǎn)染PT67細(xì)胞,48h抽提總RNA并RT-PCR檢測Tim-3的表達(dá)。PT67空細(xì)胞和pLNCX2空載體轉(zhuǎn)染的PT67細(xì)胞作對(duì)照。 研究結(jié)果 PCR、酶切及測序鑒定證實(shí)重組載體pLNCX2-Tim-3構(gòu)建成功;RT-PCR顯示pLNCX2-Tim-3在包裝細(xì)胞PT67中可有效表達(dá)Tim-3。 結(jié)論及意義 成功構(gòu)建了有效表達(dá)人Tim-3的逆轉(zhuǎn)錄病毒表達(dá)載體,為進(jìn)一步研究Tim-3表達(dá)調(diào)控機(jī)制及探索Tim-3過表達(dá)治療疾病的可行性奠定基礎(chǔ)。
[Abstract]:Tim-3 (T-cell immunoglobulin-and mucin-domain-containing molecule-3, T-cell immunoglobulin-3) is a newly discovered immunoregulatory molecule, which is specifically expressed in differentiated Th1 cells. It participates in the negative regulation of Th1 and the formation of immune tolerance, and plays an important role in the occurrence of various immune diseases. Blocking its expression can alleviate Th2. Response-induced allergic diseases are potential therapeutic targets for many immune diseases, which have attracted wide attention from scholars at home and abroad. However, there are still many problems unsolved in the study of Tim-3, especially the study of its expression regulation mechanism is still blank. The recombinant human Tim-3 promoter reporter plasmid was constructed from the gene fragment with different promoter activity, and the human Tim-3 retroviral expression vector was constructed to lay a foundation for further study on the regulation mechanism of Tim-3 expression and explore the feasibility of using Tim-3 for disease treatment.
The first part is a preliminary study on the expression pattern of PBMC Tim-3 in normal subjects.
research objective
To detect the expression of Tim-3 in human peripheral blood lymphocytes stimulated by mitogen and provide an experimental model for further study of the regulation mechanism of Tim-3 expression.
research method
After isolation of PBMC and removal of monocytes by adherence method, the suspended lymphocytes were stimulated by PHA and LPS respectively. Total RNA was extracted 24 hours later. The expression of Tim-3 was detected by RT-PCR.
Research results
RT-PCR showed that the expression of Tim-3 was up-regulated in human peripheral blood lymphocytes stimulated by PHA, but not by LPS, suggesting that the non-polarized T cells began to express Tim-3 when they were activated.
Conclusion and significance
This part establishes a cell model which can be used to study the regulation of Tim-3 expression.
The second part of human Tim-3 promoter is cloned and its activity is preliminarily studied.
research objective
To construct human Tim-3 promoter luciferase reporter plasmid and analyze its activity, which will lay a foundation for further study of the molecular mechanism of Tim-3 specific expression in Th1 cells.
research method
Prediction of 1 Tim-3 promoter region
The promoter region of human Tim-3 gene's 5 'end -2500 to +202 segment was predicted by online MatInspector software.
2 Tim-3P1 and Tim-3P2 PCR amplification
A 2325 bp-long segment of -2083~+242 was selected and cloned into Tim-3P1 (-2083~-838) and Tim-3P2 (-948~+242) based on a single Sac I cleavage site. The specific primers for Tim-3P1 and Tim-3P2 were designed and amplified by PCR using human genomic DNA as template.
Construction and identification of 3 '5' end fragment reporter plasmid containing Tim-3 gene with different lengths
Tim-3P1 PCR product, T-A cloned into pGEM-T, sequenced correctly, subcloned into the corresponding digestion site of pGL3-Basic to construct pGL3-Tim-3P1.Tim-3P2 PCR product, digested by Sac I and Xho I, recovered, inserted into pGL3-Basic polyclonal site (MCS) to construct pGL3-Tim-3P2. The promoter activity of the reported plasmid was also sequenced and identified. In the positive pGL3-Tim-3P2, SV40 enhancer sequence was used to construct Enhancer-Tim-3P2, which was identified by double enzyme digestion.
4 RT-PCR was used to detect the expression of endogenous Tim-3 in cell lines.
The logarithmic growth phase RAW264.7, B16 and COS-75 x 10~5 were collected. The total RNA of cells was extracted by Trizol and detected by RT-PCR.
5 transfection of reporter plasmid
The reported plasmids and pGL3-Basic (negative control) were transfected with pRL-TK (internal reference) to RAW264.7, B16 and COS-7 (detailed in the second part of the chart). The cells were collected 48 hours after transfection, and the crude extracts were analyzed by double luciferase assay.
6 double luciferase analysis
The relative activity of the target fragment was determined by fluorescence counting instrument. The ratio of luciferase activity M1 to luciferase activity M2 and M1/M2 in the reported plasmid was determined by fluorescence counting. Repeat 3 times.
7 Tim-3P1 and Tim-3P2 transcription factor binding site (TFBSs) analysis
The online MatInspector software is also used to analyze the possible TFBSs. in Tim-3P1 and Tim-3P2.
Research results
Prediction of 1 Tim-3 promoter region
The analysis showed that there were multiple TATA boxes, CAAT boxes and several important TFBSs in - 2500 to + 202, which might have promoter activity.
2 successfully construct human Tim-3 promoter firefly luciferase reporter plasmid.
Sequencing identification confirmed that pGL3-Tim-3P1 and pGL3-Tim-3P2 were successfully constructed, and enzyme digestion analysis confirmed that Enhancer-Tim-3P2 was successfully constructed.
3 expression of endogenous Tim-3 in different cell lines
RT-PCR detection showed that RAW264.7 and B16 expressed endogenous Tim-3 while COS-7 was not expressed.
4 validation of reporter plasmid promoter activity
Double luciferase assay confirmed that pGL3-Tim-3P1 and pGL3-Tim-3P2 had weak promoter activity, and the relative activity of pGL3-Tim-3P2 was about twice that of pGL3-Basic empty vector, and the promoter activity was cell-specific; Enhancer-Tim-3P2 promoter activity was significantly increased when introducing enhancer.
5 TFBSs analysis
The results showed that Tim-3P1 contained the following TFBSs: 1 c-Myb, Ik-1, YY1 (Yin and Yang 1, Yin and Yang 1), GATA-binding factor-3 (GATA-binding factor-3) and CHOP; 2 FOXK2, HNF-3 (hepatocyte nuclear factor-3, hepatocyte nuclear factor-3); 3 6 TATA boxes; Tim-3P2 contained the following TFBSs: 1 RFX1, p53, GATA-3, NFAT (nuclear factor of activated cells, live). Cell cycle-dependent element; cell cycle genes homology region; 2 MZF1 (Myeloid zinc finger protein-1); 3 YY1; 5 TATA boxes.
Conclusion and significance
In this part, we successfully constructed the glowworm luciferase reporter plasmid containing human Tim-3 promoter and the reporter plasmid with enhancer sequence, which laid a foundation for further study on the regulation mechanism of Tim-3 expression. Prospects.
Construction of the third part of human Tim-3 retroviral expression vector
research objective
Construction of human Tim-3 retroviral expression vector will lay a foundation for further study of the regulation mechanism of Tim-3 expression and explore the feasibility of using Tim-3 to treat diseases.
research method
A specific primer for human Tim-3 mRNA was designed and amplified by PCR using hTim-3-pGEM-T as a template. The product was cloned into the Hind III, Not I site of retroviral expression vector pLNCX2 and constructed pLNCX2-Tim-3. After being identified by PCR, enzyme digestion and sequencing, PT67 cells were transfected and the total RNA was extracted 48 hours and the expression of Tim-3 was detected by RT-PCR. PT67 cells transfected with empty carriers were used as controls.
Research results
PCR, enzyme digestion and sequencing confirmed that the recombinant vector pLNCX2-Tim-3 was constructed successfully, and RT-PCR showed that pLNCX2-Tim-3 could effectively express Tim-3 in PT67 cells.
Conclusion and significance
A retroviral expression vector expressing human Tim-3 was successfully constructed, which laid a foundation for further study on the regulation mechanism of Tim-3 expression and the feasibility of Tim-3 overexpression in the treatment of diseases.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2008
【分類號(hào)】：R392
本文編號(hào)：2216484
[Abstract]:Tim-3 (T-cell immunoglobulin-and mucin-domain-containing molecule-3, T-cell immunoglobulin-3) is a newly discovered immunoregulatory molecule, which is specifically expressed in differentiated Th1 cells. It participates in the negative regulation of Th1 and the formation of immune tolerance, and plays an important role in the occurrence of various immune diseases. Blocking its expression can alleviate Th2. Response-induced allergic diseases are potential therapeutic targets for many immune diseases, which have attracted wide attention from scholars at home and abroad. However, there are still many problems unsolved in the study of Tim-3, especially the study of its expression regulation mechanism is still blank. The recombinant human Tim-3 promoter reporter plasmid was constructed from the gene fragment with different promoter activity, and the human Tim-3 retroviral expression vector was constructed to lay a foundation for further study on the regulation mechanism of Tim-3 expression and explore the feasibility of using Tim-3 for disease treatment.
The first part is a preliminary study on the expression pattern of PBMC Tim-3 in normal subjects.
research objective
To detect the expression of Tim-3 in human peripheral blood lymphocytes stimulated by mitogen and provide an experimental model for further study of the regulation mechanism of Tim-3 expression.
research method
After isolation of PBMC and removal of monocytes by adherence method, the suspended lymphocytes were stimulated by PHA and LPS respectively. Total RNA was extracted 24 hours later. The expression of Tim-3 was detected by RT-PCR.
Research results
RT-PCR showed that the expression of Tim-3 was up-regulated in human peripheral blood lymphocytes stimulated by PHA, but not by LPS, suggesting that the non-polarized T cells began to express Tim-3 when they were activated.
Conclusion and significance
This part establishes a cell model which can be used to study the regulation of Tim-3 expression.
The second part of human Tim-3 promoter is cloned and its activity is preliminarily studied.
research objective
To construct human Tim-3 promoter luciferase reporter plasmid and analyze its activity, which will lay a foundation for further study of the molecular mechanism of Tim-3 specific expression in Th1 cells.
research method
Prediction of 1 Tim-3 promoter region
The promoter region of human Tim-3 gene's 5 'end -2500 to +202 segment was predicted by online MatInspector software.
2 Tim-3P1 and Tim-3P2 PCR amplification
A 2325 bp-long segment of -2083~+242 was selected and cloned into Tim-3P1 (-2083~-838) and Tim-3P2 (-948~+242) based on a single Sac I cleavage site. The specific primers for Tim-3P1 and Tim-3P2 were designed and amplified by PCR using human genomic DNA as template.
Construction and identification of 3 '5' end fragment reporter plasmid containing Tim-3 gene with different lengths
Tim-3P1 PCR product, T-A cloned into pGEM-T, sequenced correctly, subcloned into the corresponding digestion site of pGL3-Basic to construct pGL3-Tim-3P1.Tim-3P2 PCR product, digested by Sac I and Xho I, recovered, inserted into pGL3-Basic polyclonal site (MCS) to construct pGL3-Tim-3P2. The promoter activity of the reported plasmid was also sequenced and identified. In the positive pGL3-Tim-3P2, SV40 enhancer sequence was used to construct Enhancer-Tim-3P2, which was identified by double enzyme digestion.
4 RT-PCR was used to detect the expression of endogenous Tim-3 in cell lines.
The logarithmic growth phase RAW264.7, B16 and COS-75 x 10~5 were collected. The total RNA of cells was extracted by Trizol and detected by RT-PCR.
5 transfection of reporter plasmid
The reported plasmids and pGL3-Basic (negative control) were transfected with pRL-TK (internal reference) to RAW264.7, B16 and COS-7 (detailed in the second part of the chart). The cells were collected 48 hours after transfection, and the crude extracts were analyzed by double luciferase assay.
6 double luciferase analysis
The relative activity of the target fragment was determined by fluorescence counting instrument. The ratio of luciferase activity M1 to luciferase activity M2 and M1/M2 in the reported plasmid was determined by fluorescence counting. Repeat 3 times.
7 Tim-3P1 and Tim-3P2 transcription factor binding site (TFBSs) analysis
The online MatInspector software is also used to analyze the possible TFBSs. in Tim-3P1 and Tim-3P2.
Research results
Prediction of 1 Tim-3 promoter region
The analysis showed that there were multiple TATA boxes, CAAT boxes and several important TFBSs in - 2500 to + 202, which might have promoter activity.
2 successfully construct human Tim-3 promoter firefly luciferase reporter plasmid.
Sequencing identification confirmed that pGL3-Tim-3P1 and pGL3-Tim-3P2 were successfully constructed, and enzyme digestion analysis confirmed that Enhancer-Tim-3P2 was successfully constructed.
3 expression of endogenous Tim-3 in different cell lines
RT-PCR detection showed that RAW264.7 and B16 expressed endogenous Tim-3 while COS-7 was not expressed.
4 validation of reporter plasmid promoter activity
Double luciferase assay confirmed that pGL3-Tim-3P1 and pGL3-Tim-3P2 had weak promoter activity, and the relative activity of pGL3-Tim-3P2 was about twice that of pGL3-Basic empty vector, and the promoter activity was cell-specific; Enhancer-Tim-3P2 promoter activity was significantly increased when introducing enhancer.
5 TFBSs analysis
The results showed that Tim-3P1 contained the following TFBSs: 1 c-Myb, Ik-1, YY1 (Yin and Yang 1, Yin and Yang 1), GATA-binding factor-3 (GATA-binding factor-3) and CHOP; 2 FOXK2, HNF-3 (hepatocyte nuclear factor-3, hepatocyte nuclear factor-3); 3 6 TATA boxes; Tim-3P2 contained the following TFBSs: 1 RFX1, p53, GATA-3, NFAT (nuclear factor of activated cells, live). Cell cycle-dependent element; cell cycle genes homology region; 2 MZF1 (Myeloid zinc finger protein-1); 3 YY1; 5 TATA boxes.
Conclusion and significance
In this part, we successfully constructed the glowworm luciferase reporter plasmid containing human Tim-3 promoter and the reporter plasmid with enhancer sequence, which laid a foundation for further study on the regulation mechanism of Tim-3 expression. Prospects.
Construction of the third part of human Tim-3 retroviral expression vector
research objective
Construction of human Tim-3 retroviral expression vector will lay a foundation for further study of the regulation mechanism of Tim-3 expression and explore the feasibility of using Tim-3 to treat diseases.
research method
A specific primer for human Tim-3 mRNA was designed and amplified by PCR using hTim-3-pGEM-T as a template. The product was cloned into the Hind III, Not I site of retroviral expression vector pLNCX2 and constructed pLNCX2-Tim-3. After being identified by PCR, enzyme digestion and sequencing, PT67 cells were transfected and the total RNA was extracted 48 hours and the expression of Tim-3 was detected by RT-PCR. PT67 cells transfected with empty carriers were used as controls.
Research results
PCR, enzyme digestion and sequencing confirmed that the recombinant vector pLNCX2-Tim-3 was constructed successfully, and RT-PCR showed that pLNCX2-Tim-3 could effectively express Tim-3 in PT67 cells.
Conclusion and significance
A retroviral expression vector expressing human Tim-3 was successfully constructed, which laid a foundation for further study on the regulation mechanism of Tim-3 expression and the feasibility of Tim-3 overexpression in the treatment of diseases.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2008
【分類號(hào)】：R392
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