免疫學(xué)技術(shù)在科研中的應(yīng)用-閱讀頁
2024-08-24 02:35本頁面
【正文】 高靈敏度。 免疫細(xì)胞的檢測(cè) ? 免疫細(xì)胞的分離 ? 磁珠分離法 ? fluorescenceactivated cell sorter， FACS ? 免疫細(xì)胞功能的測(cè)定 ? T細(xì)胞 ? B細(xì)胞 磁珠分離法 ? 特異性分離所需淋巴細(xì)胞的方法。 ? 反應(yīng)管置于磁場中，鐵顆粒受磁場的吸引，攜帶有相應(yīng)細(xì)胞的磁球吸附于靠近磁鐵的管壁上。 fluorescenceactivated cell sorter， FACS T細(xì)胞鑒定及功能測(cè)定 ? 使用酶、免疫熒光標(biāo)記單抗進(jìn)行鑒定 ? 淋巴細(xì)胞轉(zhuǎn)化試驗(yàn) ? E花環(huán)形成試驗(yàn) ? 混合淋巴細(xì)胞培養(yǎng) ? CTL介導(dǎo)的細(xì)胞毒試驗(yàn) 抗原肽 —MHC分子四聚體技術(shù) tetramer ? 用生物素化的抗原肽 — MHC分子復(fù)合物與熒光標(biāo)記的親合素結(jié)合，由于 1個(gè)熒光素標(biāo)記的親合素可結(jié)合 4個(gè)生物素分子，能使 4個(gè) MHC／抗原肽復(fù)合物形成一個(gè)復(fù)合體，將該復(fù)合體標(biāo)記熒光素后，即成抗原特異性四聚體。用流式細(xì)胞術(shù)即可確定待檢標(biāo)本中抗原特異性 CTL細(xì)胞的頻率。 B細(xì)胞的鑒定及功能測(cè)定 ? 檢測(cè) B細(xì)胞分化抗原 ? 測(cè)定 B細(xì)胞產(chǎn)生抗體的能力 ? 溶血空斑試驗(yàn) ? ELISPOT 細(xì)胞因子的檢測(cè) ? 生物活性檢測(cè) ? 細(xì)胞增生或增生抑制法 ? 細(xì)胞病變抑制法 ? 趨化作用測(cè)定法 ? 免疫學(xué)檢測(cè)法 ? 分子生物學(xué)技術(shù) 基因敲除技術(shù)和轉(zhuǎn)基因動(dòng)物 ? 應(yīng)用基因同源重組，將外源有功能基因 (基因組中原先不存在、或已失活的基因 )，轉(zhuǎn)入細(xì)胞與基因組中的同源序列進(jìn)行同源重組，插人到基因組中，在細(xì)胞內(nèi)獲得表達(dá)。 ? 轉(zhuǎn)基因動(dòng)物可以作為疾病模型。 ? 轉(zhuǎn)基因動(dòng)物可作為 “ 生物反應(yīng)器 ” 生產(chǎn)藥物 。 values are mean percentages of CD44 cells within the SIINFEKL tetramerpositive population. CD8+ Memory T Cells Need Antigenspecific CD4+Thelper Cells to Achieve Tumor Protection Ovalbumin CTL epitope (SIINFEKL)specific T cells were parked in Rag/ mice for 42 days to generate mCTL and then adoptively transferred to C57BL/6 mice . Two groups of recipient C57BL/6J mice were immunized 8 days prior with 50 ug of OVT in inplete Freund’s adjuvant or 20 ug keyhole limpet hemocyanin (KLH) protein as control in inplete Freund’s adjuvant . Mice were challenged with ovalbuminexpressing tumor cells (EG7) in the scruff of the neck 1 day after adoptive transfer. Normal C57BL/6 control mice were challenged with EG7 without any treatment. CTL+ThCTL+KLHCTLThKLHControl0. 00. 20. 40. 60. 81. 0CT L + T hCT L + K L HC T LThK L HCo n tr o lTumour weight (g)Absence of tumor protection in mice without antigenspecific Thelper cells is not because of lower levels of tumor antigen (SIINFEKL)specific CD8+ T cells. mCTLs were transferred to groups of C57BL/6 mice with or without immunization 8 days prior with 50 ug of OVT or with 20 ug of KLH protein control. C57BL/6 control group was given no treatment. IFN Elispot assays were performed using the splenocytes of these mice, which were challenged in vitro with (A) 1 ug/ml SIINFEKL peptide or (B) 8ug/ml OVA Thelper peptide to determine the presence of ovalbuminspecific CD8+ CTLs and CD4+ Thelper cells. In contrast to mCTLs, eCTLs do not need T help to kill tumor. eCTLs generated from RAG/ mice were transferred at (A) day 7 or (B) day 14 to C57BL/6 mice that had been immunized 8 days earlier with 50 ug of OVT or as control 20 ug of KLH. C57BL/6 mice without any treatment were used as controls (EG7 and EL4 controls). Ovalbuminexpressing tumor cells (EG7 ) were injected under scruff of the neck of some groups of mice at (A) day 7 or (B) day 14. The parent tumor cell line EL4 that does not contain ovalbumin gene was injected into other groups of mice at day7 (A) as nonspecific tumor control. Study of the longlived memory CD8+ T cells generated in C57BL/6J mice. Ovalbumin CTL epitope (SIINFEKL)specific OT1 cells were adoptively transferred to syngeneic C57BL/6 mi
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