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具第一型人類白血球組織抗原的完全缺失、細胞異質性及亮細胞轉變特性之肉瘤狀腎細胞癌細胞株RCC52在逃避免疫監控及腫瘤進程發展上的調節作用

為了解決msi gf75災情的問題，作者謝瑾瑄 這樣論述：

指導教授推薦書口試委員會審定書誌謝.......................................................................iii中文摘要....................................................................ivAbstract...................................................................viList of abbreviations....................................

..................viiiTable of contents..........................................................xiList of Tables.............................................................xviList of Figures............................................................xviiChapter 1. Introduction.....................

...............................11.1 Major histocompatibility complex (MHC)..............................11.2 Cancer stem cells (CSCs)............................................41.3 Epithelial to mesenchymal transition (EMT)..........................81.4 Renal cell carcinoma (RCC).......

...................................91.4.1 General introduction of RCC.......................................91.4.2 Sarcomatoid RCC...................................................101.4.3 Immunohistochemical markers for diagnosis of RCC..................111.4.4 Immune escape mechanisms

of RCC...................................13Chapter 2. Total HLA class I loss in a sarcomatoid renal carcinoma cell line caused by the coexistence of distinct mutations in the two encoding beta2-microglobulin genes........152.1 Introduction........................................................15

2.2 Materials and methods...............................................162.2.1 Cell line and cell culture conditions.............................162.2.2 Cell cloning......................................................172.2.3 IFN-r.......................................................

......182.2.4 Monoclonal and polyclonal antibodies..............................182.2.5 Cytofluorometric analysis.........................................182.2.6 HLA class I genotyping............................................192.2.7 Reverse transcriptase-polymerase chain reaction (RT-

PCR)..........192.2.8 PCR and sequence analysis.........................................202.2.9 Immunohistochemistry..............................................202.2.10 Loss of heterozygosity (LOH) analysis of the b2m gene............212.3 Results.......................................

......................212.3.1 HLA class I and class II antigen expression on the six RCC cell lines analyzes...212.3.2 Intracellular expression of HLA class I heavy chain, b2m, TAP subunits and LMP subunits in the RCC cell lines analyzed..................................................232.3.3

Expression of b2m, TAP and LMP mRNAs in the RCC cell lines analyzed..............232.3.4 Identification of b2m gene mutations in the RCC52 cell line........242.3.5 Distinct b2m gene mutations carried by subpopulations in the RCC52 cell line.....242.3.6 Lack of b2m and HLA class I antige

n expression in the sarcomatoid component in the tumor lesion from which the RCC52 cell line was originated...............................252.3.7 Detection of LOH at STR D15S-209 of the b2m gene only in RCC52 epithelioid sublines....................................................................

.............262.4 Discussion........................................................27Chapter 3. Co-existence of epithelioid and fibroblastoid subsets in a sarcomatoid renal carcinoma cell line revealed by clonal studies...........................................323.1 Introduction............

..........................................323.2 Materials and methods.............................................333.2.1 Patient tumor specimens and tissue processing for cell culture....333.2.2 Growth curves.....................................................353.2.3 Monoclonal antibod

ies and cytofluorometric analysis...............363.2.4 Cell migration and invasion assays................................373.2.5 ELISA.............................................................383.2.6 Anchorage-independent colony-formation assay......................383.2.7 Protein ex

traction and western blot analysis......................393.2.8 Xenotransplantation in immunodeficient mice.......................393.2.9 Immunohistochemistry and immunocytochemistry......................403.2.10 Statistical analysis..............................................413.3 Res

ults...........................................................423.3.1 Establishment of the RCC52 cell line..............................423.3.2 In vitro growth characteristics of parental and clonal sublines of the RCC52 cell line...............................................................

......................423.3.3 Immunophenotyping of parental and clonal sublines of the RCC52 cell line.........433.3.4 Differences between the epithelioid and fibroblastoid sublines were further compared by cytofluorometric analysis....................................................443.3.5

Difference in cell migration and invasion ability between the epithelioid and fibroblastoid clonal sublines............................................................453.3.6 Differential release of TGF-b1 by epithelioid and fibroblastoid subsets..........453.3.7 Difference in CD105 expressio

n between epithelioid and fibroblastoid clonal sublines.................................................................................463.3.8 Anchorage-independent colony-forming ability of parental RCC52, epithelioid and fibroblastoid sublines...................................................

................463.3.9 Difference in Grp78 expression between the epithelioid and fibroblastoid clonal sublines.................................................................................473.3.10 Xenotransplantability in immunodeficient mice and characterization of cell cultures re-estab

lished from the xenografts..............................................473.3.11 Immunohistochemistry of xenografts resulting from subcutaneous injection of parental and clonal sublines of the RCC52 cell line......................................483.4 Discussion................................

........................50Chapter 4. Differential expression of CD44 and CD24 discriminates the epithelioid versus fibroblastoid subset of the sarcomatoid renal carcinoma RCC52 cell line ensuing common and divergent functional roles in tumor progression...............................................

...............................584.1 Introduction......................................................584.2 Materials and methods...................................................................614.2.1 Cell lines and culture conditions.................................614.2.2 Growth cu

rves.....................................................614.2.3 Cytofluorometric Analysis.........................................614.2.4 Cell migration and invasion assays................................634.2.5 Anchorage-independent colony-formation assay......................644.2.6 X

enotransplantation in immunodeficient mice.......................644.2.7 Immunohistochemistry..............................................654.2.8 Statistical analysis..............................................664.3 Results...........................................................664.3.

1 Differential expression of CD44 and CD24 expressed on a panel of RCC cell lines..664.3.2 Differential expression of CD44 and CD24 on the epithelioid and fibroblastoid sublines.................................................................................674.3.3 In vitro growth and immun

ophenotypic characterization of the two sorted RCC52 subsets..................................................................................674.3.4 Migration and invasion ability of the two sorted RCC52 subsets...................684.3.5 Differential release of TGF-β1 by the two sorted RCC52

subsets..................694.3.6 Anchorage-independent colony forming ability of the two sorted RCC52 subsets.....694.3.7 Xenotransplantation of the two sorted RCC52 subsets...............704.3.8 Histochemical patterns of xenografts resulting from injection of cultured cells of the two sort

ed RCC52 subsets.............................................................704.3.9 Expression of surface CD105 on the two sorted RCC52 subsets.......724.4 Discussion........................................................72Chapter 5. Conclusions and Future perspectives.......................

.....79References................................................................82Tables....................................................................96 Table 1. The cell surface phenotype of CSCs identified in human cancer...96 Table 2. Immunohistochemical markers in the diagnosis of RCC....

.........97Table 3. Diagnosis and clinical information of patients from whom the six RCC cell lines were established.........................................................................98Table 4. RT-PCR primers used in this study................................99Table 5. Genetic abnormalities in

the β2m locus of the RCC52 epithelioid and fibroblastoid clonal sublines...........................................................100Table 6. Expression of selected surface and cytoplasmic markers by RCC52 cells as determined by cytofluorometric analysis............................................

.....101Table 7. Surface and cytoplasmic expression of selected antigens on the RCC52 parental and clonal sublines as determined by cytofluorometric analysis..............................103Table 8. Surface and cytoplasmic expression of selected antigens on the RCC52 parental and CD44/CD24 sorted ce

lls of determined by cytofluorometric analysis.......................105Table 9. Comparative results of immunophenotypical and biological properties between the RCC52 epithelioid and fibroblastoid subsets using cytofluorometrically sorted cells vs. clonally derived sublines..........................

.....................................107Figures..................................................................108 Figure 1. Differential surface HLA class I and HLA class II antigen expression on the RCC cell lines..............................................................................108Fi

gure 2. HLA class I and APM components expression by RCC cell lines....109Figure 3. Upregulation by IFN-r of b2m, LMP2, LMP7, TAP1 and TAP2 transcripts in the RCC cell lines and in the control cell line.................................................110Figure 4. Identification of two frame-shift mu

tations in the b2m gene in RCC52 cells....111Figure 5. Monolayer culture morphology of the RCC52 cell line and its representative clonal sublines.........................................................................113Figure 6. Sequence abnormalities of the b2m genes of the RCC52 cell line and it

s representative clonal sublines..........................................................114Figure 7. H&;E and immunohistochemical staining of a RCC lesion from which the RCC52 cell line was established....................................................................115Figure 8. LOH analysis of

chromosome 15 STR markers D15S-126 and D15S-209 flanking the b2m gene on the RCC52 cell line, epithelioid and fibroblastoid sublines.....................116Figure 9. Histopathology of tumor and in vitro live monolayer culture of RCC52 parental and fibroblastoid clonal sublines.......................

................................117Figure 10. In vitro growth of RCC52 parental and fibroblastoid clonal sublines..........118Figure 11. In vitro migration/invasion potential of two types of RCC52 clonal sublines..119Figure 12. TGF-b1 secretion levels of two types of RCC52 clonal sublines...........

.....120Figure 13. Cytofluorometric analysis of surface CD105 expression and anchorage- independent cell growth of RCC52 clonal sublines........................................121Figure 14. Western blot of Grp78 in RCC52 clonal sublines................122Figure 15. Xenotransplantation of RCC52 paren

tal and clonal sublines.....123Figure 16. Immunostaining of PAX2 on tumor sections......................124Figure 17. Two color cytofluorometric analysis on six different histologic RCC cell lines...................................................................................125Figure 18. Two col

or cytofluorometric analysis on RCC52 cells and the clonal sublines...126Figure 19. Morphology and in vitro growth patterns of sorted RCC52 cells................127Figure 20. In vitro migration and invasion ability of the sorted RCC52 cells............128Figure 21. TGF-b1 secretion levels of RCC52 s

orted cells.................129Figure 22. Anchorage-independent cell colony-forming-ability in RCC52 sorted cells......130Figure 23. Xenotransplantation results of CD44/CD24 sorted RCC52 cells..................131Figure 24. Histochemistry of the xenografts in SCID/NOD mice resulting from injection w

ith CD44/CD24 sorted RCC52 cells............................................................132Figure 25. Immunostaining of PAX2 reactivity on tumor sections of the representative xenografts resulting from sorted RCC52 cells............................................133Figure 26. Cytofluorometric a

nalysis of surface CD105 expression on RCC52 sorted cells..134Appendix I. Microarray results of the RCC52 clonal sublines..............135Appendix II. Publications................................................137

以即時定量PCR技術偵測微衛星DNA量的變異的可行性之研究

為了解決msi gf75災情的問題，作者康佳瑜 這樣論述：

摘要對偶基因不平衡所造成的LOH（loss of heterozygousity）是腫瘤發生過程中獨特的遺傳變異機制之一。Gene Scan偵測方式會受限於異結合比例，微衛星定量分析（QuMA）不僅能克服此問題，還能區分變異是放大或缺失。本實驗欲以QuMA建立偵測遺傳變異的系統，並與Gene Scan比較是否具一致性，進而取代之。沿用Gene Scan篩選的一級標記D17S849（17p）、D8S505（8q）和二級標記D16S3091（16q），對肝癌組織、血漿、血球進行相對定量，所用的內在校正有兩種：參考基因（β-actin）及螢光測量法(Fluorometry)所得DNA原始濃度。結果

如下所述，（1）組織－β-actin為內在校正時，QuMA和Gene Scan結果相同的比例為：82.1％（D17S）、90％（D8S）、75％（D16S），兩種方法以 Kappa一致性分析，確實呈現高度符合的結果（p＝0.01、p＝1.6×10-4、p＝0.02）。Fluorometry為內在校正部分：60.7％（D17S）、53.3％（D8S）、66.7％（D16S）。（2）血漿－β-actin部分結果一樣的比例是33.3％（D17S）、65.2％（D8S）、46.7％（D16S）。Fluorometry部分35％（D17S）、27.3％（D8S）、66.7％（D16S）。本實驗發現β-a

ctin當內在校正比較精確，Fluorometry不適合可能是未經過PCR反應過程。血漿結果差異甚大，可能是Gene Scan測量血漿微衛星DNA不是很靈敏，因為有許多檢體皆偵測不到，而且血漿半衰期很短，導致符合比例不高。（3）血球－QuMA測得肝癌與正常人的相對量進行判別分析，得到組別正確分類的機率是91.9%，可見正常人與癌症可以相對量區分。進一步分析三組微衛星以QuMA和Gene Scan檢測後，整體存活率及無病存活率皆無顯著關係，原因是當初Gene Scan僅根據組織、血漿中LOH的比例認定這三組標記，並沒有將預後的關係包含在內。本實驗還發現， Fluorometry測得的血漿原始濃度

和轉移有顯著關係（p =0.0048）。本實驗證明QuMA確實不受限異結合比例、可快速地進行篩檢、操作又比Gene Scan方便，希望在未來能運用於癌症的早期診斷與預後評估。