【作者】 黄江生；

【导师】 张阳德；

【作者基本信息】 中南大学 ， 外科学， 2009， 博士

【摘要】 第一部分癌干细胞及其标记物研究目的研究胆囊腺癌、癌旁上皮、腺瘤性息肉和慢性胆囊炎组织中前列腺干细胞抗原（prostate stem cell antigen,PSCA）、Oct-4干细胞抗原（Oct-4）、CD24和CD_44V6表达水平,探讨其临床病理意义及在胆囊腺癌发生发展中的作用及作用机理。方法108例胆囊腺癌、46例癌旁组织、15例腺瘤性息肉和35例慢性胆囊炎手术切除标本经4%甲醛固定后常规制作石蜡包埋切片,PSCA、Oct-4、CD24和CD_44V6染色方法均为Envision^TM免疫组化二步法。结果（1）108例胆囊腺癌PSCA、Oct-4、CD24和CD_44V6表达阳性病例分别为59例（54.6%）、60例（55.6%）、57例（52.8%）和60例（55.6%）,其评分值分别为2.09±1.94、2.06±1.86,2.43±1.65和2.52±1.60;46例癌旁组织PSCA、Oct-4、CD24和CD_44V6表达阳性病例分别为9例（19.6%）、10例（22.5%）、8例（17.4%）和9例（19.6%）,其评分值分别为0.82±1.11、0.85±1.02、0.78±1.36和0.82±1.39;15例腺瘤性息肉PSCA、Oct-4、CD24和CD_44V6表达阳性病例分别为4例（26.7%）、3例（20.0%）、3例（20.0%）和4例（26.7%）,其评分值分别为0.74±1.25、0.69±1.32、0.72±1.35和0.78±1.28;35例慢性胆囊炎PSCA、Oct-4、CD24和CD_44V6表达阳性病例分别为5例（14.3%）、5例（14.3%）、4例（11.4%）和6例（17.1%）,其评分值分别为0.68±1.21、0.67±0.95、0.54±1.44和0.58±1.46;PSCA、Oct-4、CD24或（和）CD_44V6表达阳性的癌旁组织、腺瘤性息肉和慢性胆囊炎胆囊上皮均呈中度至重度不典型增生;胆囊腺癌PSCA、Oct-4、CD24和CD_44V6表达阳性率及其评分明显高于癌旁上皮(PSCA,x²=10.09,P<0.01,τ=4.23,P.x²<0.01 Oct-4,x²=14.88,P<0.01,τ=4.32,P<0.01;CD24,x²=16.58,P<0.01,τ=5.89,P<0.01;CD_44V6,x²=16.90,P<0.01,τ=6.07,P<0.01)、腺瘤性息肉(PSCA,x²=4.12,P<0.05,τ=2.60,P<0.05;Oct-4,x²=6.66,P<0.01,τ=280,P<0.01;CD24,x²=5.66,P<0.05,τ=3.78 P<0.01;CD_44V6,X²=4.12,P<0.05,τ=4.05,P<0.01)和慢性胆囊炎(PSCA,X²=17.40,P<0.01,τ=4.15,P<0.01;Oct-4,x²=18.16,P<0.01,τ=4.21,P<0.01;CD24,x²=18.48,P<0.01,τ=6.10,P<0.01;CD_44V6,x²=15.69,τ=6.26,P（0.01）。（2）腺瘤癌变或高分化腺癌、肿块最大径<2cm、无淋巴结转移及侵犯胆囊外周围组织器官病例PSCA、Oct-4、CD24和CD_44V6表达阳性率及其评分明显低于低分化腺癌、肿块最大径≥2cm、淋巴结转移及侵犯胆囊外周围组织器官病例,差异均有显著性或高度显著性（P<0.05或P<0.01）。（3）胆囊腺癌中PSCA、Oct-4、CD24和CD_44V6表达水平相互之间具有高度一致性（P<0.01）,其评分值相互之间存在密切正相关(PSCAvs Oct-4,r=0.44,P<0.01;PSCA vs CD24,r=0.36,P<0.01;PCSA vsCD_44V6,r=0.49;P<0.01,Oct-4 vs CD24,r=0.51,P<0.01;Oct-4 vsCD_44V6,r=0.39,P<0.01;CD24 vs CD_44V6,r=0.48,P<0.01)。结论PSCA、Oct-4、CD24和CD_44V6作为癌干细胞标记物在胆囊腺癌发生、进展、转移及侵袭潜力方面具有重要作用,阳性或高水平表达者临床预后不良;癌组织中存在的癌干细胞有可能在胆囊腺癌发生发展中起重要作用,且影响胆囊腺癌临床生物学行为及其预后;检测胆囊良性病变组织中PSCA、Oct-4、CD24和CD_44V6 4种癌干细胞标记物表达水平对预防和早期发现胆囊癌可能具有重要临床应用价值。第二部分胆囊腺癌转移和侵袭调控机制研究第一章胆囊良恶性病变微血管和淋巴管计数及其意义目的研究胆囊腺癌、癌旁组织、腺瘤性息肉和慢性胆囊炎组织中微血管（MV）和淋巴管（LV）计数及其临床病理意义。方法108例胆囊腺癌、46例癌旁组织、15例腺瘤性息肉和35例慢性胆囊炎外科手术切除标本经4%甲醛固定后常规制作石蜡包埋切片,MV和LV染色方法均为S-P免疫组化法,低倍镜下选择MV和LV分布最丰富的10个区域,高倍镜下计数10个区域内MV和LV数,取其均值为该病例计数值。结果108例胆囊腺癌MV和LV计数均值（64.2±11.6,11.4±5.2）明显高于癌旁组织（28.2±12.4,τ=17.6,P<0.01;6.8±6.2,τ=4.63,P<0.01）、腺瘤性息肉（32.4±11.8,τ=9.94,P<0.01;5.6±2.9,τ=4.17,P<0.01）和慢性胆囊炎（22.2±11.3,τ=18.26,P<0.01;5.1±3.9,τ=6.63,P<0.01）。腺瘤癌变或高分化腺癌、肿块最大径<2cm、无淋巴结转移及未侵犯周围组织器官病例MV和LV计数均值明显低于低分化腺癌、肿块最大径≥2cm、淋巴结转移及侵犯周围组织器官病例（P<0.01）。胆囊腺癌中MV计数与LV计数呈密切正相关（r=0.38,P<0.01）。结论MV和LV计数可能是反映胆囊腺癌进展,临床生物学行为及预后的标记物第二章转移抑制调控基因的研究目的研究胆囊腺癌、癌旁组织、腺瘤性息肉和慢性胆囊炎组织中KAI-1mRNA、Kiss-1mRNA、MTA1和CD146表达水平及其临床病理意义。方法108例胆囊腺癌、46例癌旁组织、15例腺瘤性息肉和35例慢性胆囊炎组织经4%甲醛固定后常规制作石蜡包埋切片,KAL-1mRNA和Kiss-1mRNA染色方法为原位杂交染色方法,MTA1和CD146染色方法为Envision^TM免疫组化二步法。结果（1）胆囊腺癌KAL-1mRNA和Kiss-1mRNA表达阳性率（51.9%,53.7%）明显地低于癌旁组织（KAL-1mRNA,80.4%,x²=11.02,P<0.01;Kiss-1mRNA,82.6%,x²=11.48,P<0.01）腺瘤性息肉（KAL-1mRNA,86.7%,x²=6.48,P<0.05;Kiss-1mRNA,86.7%,x²=5.86P<0.05）和慢性胆囊炎(KAL-1mRNA,85.7%,x²=12.64,P<0.01;Kiss-1mRNA,85.7%,x²²=11.44,P<0.01);胆囊腺癌MTA1和CD146表达阳性率（48.1%,53.7%）明显高于癌旁组织（MTA1,26.1%,x²=6.46,P<0.05;CD146,30.4%,x²=7.02,P<0.01）,腺瘤性息肉（MTA1,20.0%,x²=4.22,P<0.05;CD146,20.0%,x²=8.59,P<0.01）和慢性胆囊炎（MTA1,8.6%,x²=13.31,P<0.01,CD146,5.7%,x²=25.00,P<0.01）。（2）腺瘤癌变或高分化腺癌、肿块最大径<2cm、无淋巴结转移及未侵犯周围组织器官病例KAL-1mRNA、Kiss-1mRNA表达阳性率明显高于低分化腺癌、肿块最大径≥2cm、淋巴结转移及侵犯周围组织器官病例（P<0.05或P<0.01）,但MTA1和CD146在胆囊腺癌中表达与KAL-1mRNA、Kiss-1mRNA表达相反（P<0.05或P<0.01）。（3）KAL-1mRNA和/或Kiss-1mRNA表达阳性胆囊腺癌MV和LV计数明显低于阴性表达病例（P<0.01）;MTA1和/或CD146表达阳性胆囊腺癌MV和LV计数明显高于阴性表达病例（P<0.01）。结论KAL-1mRNA、Kiss-1mRNA、MTA1和CD146表达水平均为反映胆囊腺癌发生、进展,临床生物学行为及预后的重要分子标记物,均为重要的调控胆囊癌等恶性肿瘤转移和侵袭能力的重要指标,其作用可能与调控肿瘤血管生成和淋巴生成有关。更多还原

【Abstract】 Part One Cancer Stem Cells and Their MarkersObjective To study the expression levels of prostate stem cell antigen（PSCA）,Oct-4,CD24 and CD44V6 in the gallbladder adenocarcinoma,perirumoral tissues,adenomatous polyp,and chronic cholecystitis,and to detect their clinicopathologic significances in gallbladder adenocarcinoma.Methods Immunohistocemical method of EnVision^TM（two steps）for PSCA,Oct-4,CD24 and CD44V6 was used in the routinely paraffin-embedded sections（after 4%formaldehyde fixing）of specimens from gallbladder adenocarcinoma（n=108）,peritumoral tissues（n=46）, adenomatous polyp（n=15）,and chronic cholecystitis（n=35）Results（1） In the 108 cases of gallbladder adenocarcinoma,PSCA, Oct-4,CD24 and CD44V6 positive cases were respectively 59（54.6%）,60 （55.6%）,60（55.6）,57（52.8%）,and 60（55.6%）,and their scores were respectively2.09±1.94,2.06±1.86,2.43±1.65,and 2.52±1.60.In the 46 cases of perirumoral tissues,PSCA,Oct-4,CD24 and CD44V6 positive cases were respectively 9（19.6%）;10（22.5%）;8（17.4%）,and 9 （19.6%）,their scores were respectively 0.82±1.11 0.85±1.02 0.78±1.36 and 0.82±1.39.In the 15 cases of adenomatous polyp,PSCA,Oct-4,CD24 and CD44V6 positive cases were respectively 4（26.7%）,3（20%）,and 4 （26.7%） and their scores were respectively 0.74±1.25,0.69±1.32, 0.72±1.35,and 0.78±1.28 In the 35 cases of chronic cholecystitis,PSCA,oct-4,CD24,and.CD44V6 positive cases were respectively 5（14.3%） 5（14.3%） 4（14.4%） and 6（17.1%）,and their scores were respectively 0.68±1.21,0.67±0.95,0.54±1.44,and 0.58±1.46, The gallbladder epithelium of PSCA,Oct-4,CD24 and CD44V6 positive cases all showed middle to severely-atypical hyperplasia.The positive rates of PSCA,ct-4,CD24 and CD44V6 expressions and their scores in gallbladder adenocarcinoma were significantly higher than those in peritumoral tissues（PSCA,x²=10.09,P＜0.01,τ=4.23,P＜0.01;Oct-4, x²=14.88,P＜0.01,τ=4.32,P＜0.01;CD24,x²=16.58,p＜0.01,t=5.89, P＜0.01;CD44V6,x²=16.90,P＜0.01,τ=6.07,P＜0.01）,adenomatous polyp （PSCA,x²=4.12,P＜0.05;τ=2.60P＜0.05 Oct-4,x²=6.66,p＜0.01,τ=2.80, P＜0.01;CD24,x²=5.66,P＜0.05,τ=3.78,P＜0.01;CD44V6,x²=4.12, P＜0.05,τ=4.05,P＜0.01）,and chronic cholecystitis（PSCA,x²=17.40, P＜0.01,τ=6.10,P＜0.01;Oct-4,x²=18.16,p＜0.01,τ=4.21,P＜0.01;CD24, x²=18.48,P＜0.01,τ=6.10,P＜0.01;CD44v6,x²=15.69,P＜0.01,τ=6.62, P＜0.01）.（2）The positive rates of PSCA,Oct-4,CD24 and CD44V6 expressions and their scores were significantly lower in the cases of adenomatous canceration or well-differentiated adenocarcinoma,maximal diameter of mass＜2cm,no-metastasis of lymph node,and no-invasion of regional tissues than those in the ones of pooly-differentiated adenocarcinoma, maximal diameter of mass≥2cm,metastasis of lymph node and invasion of regional tissues（P＜0.05,or P＜0.01）.（3）Highly consistencies in PSCA,Oct-4,CD24 and CD44V6 expressions were found in gallbladder adenocarcinoma（P＜0.01）,and significantly positive correlations were found among their scores（PSCA vs Oct-4,r=0.44,P＜0.01;PSCA vs CD24,r=0.36,P＜0.01;PSCA vs CD44V6,r=0.49,P＜0.01;Oct-4 vs CD24,r=0.51,P＜0.01;Oct-4 vs CD44V6,r=0.39,P＜0.01;CD24 vs CD44v6,r=0.48,P＜0.01）.Conclusions PSCA,Oct-4,CD24 and CD44v6,as markers of cancer stem cells,played an important role in the carcinogenesis, progression,metastatic potency,invasive capability of gallbladder adenocarcinoma,and positive or high expression ones had poor prognostis The cancer stem cells in the cancer tissues might play an important role in the genesis,progression,biological behavior and prognosis of gallbladder adenocarcinoma.Detecting the PSCA,Oct-4,CD24 and CD44V6 levels in the benign lesions of gallbladder might have significant clinical values for the prevention and early-finding of gallbladder adenocarcinoma. Part Two the Controlling Mechanism of Metastasis and Invasion of Gallbladder AdenocarcinomaChapter One Microvessels counts and lymph vessels counts and their significances in benign and malignant lesions of gallbladderObjective To study the microvessel（MV）counts,and lymph vessel （LV）counts in gallbladder adenocarcinoma,peritumoral tissues, adenomatous polyp,and chronic cholecystitis,and to detect their clinicopathologic significances in gallbladder adenocarcinoma.Methods Streptavidin-peroxidase immunohistochemical methods for MV and LV was used in the routinely paraffin-embedded sections（after 4%formaldehyde fixing）of specimens from gallbladder adenocarcinoma（n=108）,peritumoral tissues（n=46）,adenomatous polyp（n=15）,and chronic cholecystitis（n=35）.Selected ten regions tha MV and LV were most dense under the low-power field,and then counted the numbers of MV and LV of the ten regions under the high-power microscope;the average of the numbers was the counts of MV and LV of the case.Results The counts of MV and LV（64.2±11.6,11.4±5.2）in gallbladder adenocarcinoma were significantly higher than those in peritumoral tissues（28.2±12.4,τ=17.6,P＜0.01;6.8±6.2,τ=4.63,P＜0.01）, adenomat-ous polyp（32.4±11.8,τ=9.94,P＜0.01;5.6±2.9,τ=4.17, P＜0.01）,and chronic cholecystitis（22.2±11.3,τ=18.26,P＜0.01;5.1±3.9, τ=6.63,P＜0.01）.The counts of MV and LV were significantly lower in the cases of adenomatous canceration or well-differentiated adenocarcinoma,maximal diameter of mass＜2cm,no-metastasis of lymph node,and no-invasion of regional tissues than those in the ones of poorly-differentiated adenocarcinoma,maximal diameter of mass≥2cm, metastasis of lymph node,and invasion of regioonal tissues（P＜0.01）. Significantly positive correlation was found between MV counts and LV counts in gallbladder adenocarcinoma（r=0.38,p＜0.01）.Conclusions MV counts and LV counts might be important biological markers reflecting the progression,clinical behavior and prognosis of gallbladder adenocarcinoma.Chapter Two Study of metastatic controlling genesObjective To study the expressions of KAI-1mRNA, Kiss-1mRNA,MTA1 and CD146 in gallbladder adenocarcinoma,peritumoral tissues,adenomatous polyp,and chronic cholecystitis,and to detect their clinico- pathologic significances in gallbladder adenocarcinoma.Methods In situ hybridization for staining KAI-1mRNA and Kiss-1mRNA,and immunohistochemical method of EnVision^TM（two steps）for MTA1 and CD146 were used in the routinely paraffin-embedded sections（after 4%formaldehyde fixing） of specimens from gallbladder adenocarcinoma（n=108）,peritumoral tissues（n=46）,adenomatous polyp（n=15）,and chronic cholecystitis（n=35）.Results The positive rates of KAI-1mRNA and Kiss-1mRNA （51.9%,53.7%）were significantly lower in gallbladder adenocarcinoma than those in peritumoral tissues（KAI-1mRNA,80.4%,x²=11.02, P＜0.01;Kiss-1mRNA,82.6%,x²=11.48,P＜0.01）,adenomatous polyp（KAI-1mRNA,86.7%,x²=6.48,P＜0.05;Kiss-1mRNA,86.7%,x 2=5.86,P＜0.05）;and chronic cholecystitis（KAI-1mRNA 85.7%,x²=12.64, P＜0.01;Kiss-1mRNA,85.7%,x²=11.44,P＜0.01）.The positive rates of MTA1 and CD146（48.1%,53.7%）were significantly higher in gallbladder adenocarcinoma than those in peritumoral tissues（MTA1,26.1%,x 2=6.46,P＜0.05;CD146,30.4%,x²=7.02,P＜0.01）,ademomatous polyp（MTA,20.0%,x²=4.22,P＜0.05;CD146,20.0%,x²=8.59%, P＜0.01）;and chronic cholecystitis（MTA1,8.6%,x²=13.31,P＜0.01; CD146,5.7%,x²=25.00,P＜0.01）.The positive rates of KAI-1mRNA and kiss-1mRNA were significantly higher in cases with adenomafous canceration or well differentiated adenocarcinoma,maximal diameter of regional tissues＜2cm,no-metastasis of lgmph node and no-invasion of regional tissues than in those with poorly-differentiated adenocarcinoma or mucinous adenocarcinoma,maximal diameter of mass≥2cm, metastasis of lymph node and invasion of regional tissues（P＜0.05 or P＜0.01）,while MTA and CD146 were on the contrary（P＜0.05 or P＜0.01）.The counts of MV and LV in gallbladda adenocarcinoma were significantly lower in KAI-1mRNA and/on kiss-1mRNA positive cases than those in negative cases（P＜0.01）,while the counts of MV and LV in gallbladder adenocarcinoma were significantly higher in MTA1,and/or CD146 positive cases than those in negative cases（P＜0.01）.Conclusions KAI-1mRNA,kiss-1mRNA,MTA1 and CD146 might be important biological markers reflecting the genesis,progression,clinic biological behavior and prognosis of gallbladder adenocarcinoma,and important markers controlling the metastasis and invasion abilities of gallbladder adenocarcinoma.Their controlling impact might correlate with their controlliny of the angiogenesis and lymphangiolgenesis of tumors.更多还原