【作者】 李凯；

【导师】 王春友；

【作者基本信息】 华中科技大学 ， 外科学， 2007， 博士

【摘要】 胰腺癌是预后最为恶劣的消化系统恶性肿瘤之一,90%的患者就诊时已属进展期,治疗手段单一,手术切除率低,5年生存率不超过5%。而胰腺癌早期即可发生区域淋巴结转移,是影响患者预后的重要因素。因此,对胰腺癌淋巴结转移机制及干预手段的深入研究具有重要的意义。VEGF-C基因是1996年被克隆定位于人染色体4q34上的一个新基因。近年来的大量研究结果显示,其与肿瘤淋巴结转移高度特异性相关。目前认为,VEGF-C通过结合淋巴管内皮细胞上表达的受体VEGFR-3促进肿瘤“淋巴管生成”是肿瘤淋巴结转移的关键机制之一。但实验研究证实,沉默VEGFR-3的表达以抑制肿瘤“淋巴管生成”对已转移至淋巴结的肿瘤细胞并无抑制作用。因此,对就诊时多已发生淋巴结转移的胰腺癌患者来说,抑制肿瘤“淋巴管生成”的效果可能并不乐观。最近,有研究结果提示肿瘤细胞VEGF-C的异常表达与肿瘤细胞寄生部位的局部微环境关系密切。进一步通过免疫组织化学染色的方法,在乳腺癌原位种植瘤模型中发现存在部分转移至淋巴管内的乳腺癌细胞VEGF-C阳性表达增多的情况;而对人胰腺癌标本中也发现,转移至淋巴结的胰腺癌细胞VEGF-C染色阳性率明显高于原发灶的有趣现象。那么,在淋巴结转移灶局部微环境的影响下,胰腺癌细胞VEGF-C表达是否存在器官差异性呢?如果存在,这种差异性对胰腺癌细胞淋巴结转移又发挥何种作用,能否为我们提供一个抑制胰腺癌细胞淋巴结转移的新思路呢?通过对VEGF-C在胰腺癌淋巴结转移中作用的进一步认识,其能否成为判断胰腺癌预后的一种辅助指标呢?为此,我们进行了以下三个部分的研究。第一部分VEGF-C在胰腺癌细胞种植瘤模型原位灶与淋巴结转移灶中表达的差异性目的:研究胰腺癌PANC-1细胞在原位种植瘤模型原位灶与淋巴结转移灶中VEGF-C表达的差异性。方法:建立胰腺癌PANC-1细胞原位种植瘤模型,原代分离培养原位灶与淋巴结转移灶中肿瘤细胞,通过RT-PCR、Western Blot、ELISA等方法检测其VEGF-C的表达情况。结果:胰腺癌PANC-1细胞原位种植瘤模型成瘤率100.0%,自发性淋巴结转移发生率为62.5%,自发性肝转移发生率为25.0%,未见肺转移。将原代分离培养的原位灶与淋巴结转移灶中胰腺癌细胞分别命名为PANC-1-PRO和PANC-1-LN细胞。两细胞中VEGF-C基因mRNA表达水平分别为0.61±0.15与0.87±0.11,细胞中的VEGF-C蛋白表达水平分别为0.65±0.17与0.88±0.09,细胞培养液上清液中VEGF-C蛋白表达含量分别为(1403.67±128.15)pg/ml与(1682.37±156.73)pg/ml,PANC-1-LN细胞VEGF-C的表达水平均明显高于PANC-1-PRO细胞,差异有统计学意义(t=3.1255、2.9062、3.0782,P=0.0141、0.0197、0.0152)。结论:原位种植瘤模型自发性淋巴结转移灶中胰腺癌PANC-1细胞的VEGF-C表达水平显著高于原位灶中肿瘤细胞,胰腺癌细胞的VEGF-C表达存在器官差异性。第二部分VEGF-C特异性反义寡核苷酸对淋巴结转移灶胰腺癌细胞凋亡的影响目的:研究VEGF-C特异性反义寡核苷酸对原位灶与淋巴结转移灶中胰腺癌细胞凋亡的影响。方法:建立胰腺癌PANC-1细胞原位种植瘤模型,原代分离培养原位灶与淋巴结转移灶中肿瘤细胞(PANC-1-PRO和PANC-1-LN细胞),分为空白对照、错义寡核苷酸、反义寡核苷酸3个处理组。应用RT-PCR、Western Blot、ELISA、流式细胞术、TUNEL等方法检测VEGF-C反义寡核苷酸的体内外转染对其表达水平以及对胰腺癌细胞凋亡的影响。结果:体外转染后,反义组PANC-1-PRO和PANC-1-LN细胞中VEGF-C的mRNA、蛋白表达水平及细胞培养液上清液中蛋白表达含量均较空白、错义组显著降低(P<0.01)。而3组PANC-1-PRO和PANC-1-LN细胞中凋亡相关基因bcl-2的mRNA表达水平分别为0.67±0.12、0.69±0.14、0.61±0.11和0.56±0.16、0.59±0.18、0.27±0.17,仅PANC-1-LN细胞的反义组bcl-2表达水平较前2组明显下调(t=3.0428、3.1659,P=0.0124、0.0101)。PANC-1-PRO细胞转染后,3组的早期细胞凋亡率分别为(3.51±1.38)%、(4.79±2.16)%、(5.33±2.18)%,差异无统计学意义(t=1.7279、0.4310,P=0.1147、0.6756);而PANC-1-LN细胞3组早期细胞凋亡率分别为(2.83±1.01)%、(4.98±2.05)%、(13.22±2.17)%,反义组细胞凋亡率显著上升,差异有显著统计学意义(t=10.6329、6.7613,P=0.0000、0.0000)。体内转染后,反义组种植瘤模型血清中VEGF-C蛋白含量较空白、错义组显著降低(P<0.05)。3组模型瘤体体积、淋巴结转移发生率及转移淋巴结数的差异均无统计学意义(P>0.05)。3组原位灶胰腺癌细胞凋亡率分别为(1.29±0.53)%、(1.98±0.77)%、(2.01±0.80)%,组间差异均无统计学意义(t=2.1221、0.0764,P=0.0522、0.9402);而3组淋巴结转移灶胰腺癌细胞平均凋亡率分别为(1.78±0.49)%、(1.96±0.68)%、(4.38±1.02)%,反义组细胞凋亡率显著上升,差异有显著统计学意义(t=6.4987、5.2301,P=0.0000、0.0001)。结论:抑制淋巴结转移灶中胰腺癌细胞VEGF-C的表达可促进淋巴结转移灶中胰腺癌细胞的凋亡,和bcl-2表达下调有关;但对原发灶胰腺癌细胞无明显影响。VEGF-C基因的作用机制也存在器官差异性,是胰腺癌淋巴结转移的机制之一。第三部分无法切除胰腺癌患者血清VEGF-C蛋白含量与预后相关性的研究目的:研究无法切除胰腺癌患者血清中VEGF-C蛋白含量与预后的关系。方法:选择华中科技大学同济医学院附属协和医院胰腺外科中心2004年12月～2005年8月间无法切除的局部晚期胰腺癌病例35例,记录入组时的KPS评分、CT资料的肿瘤最大直径及血清CA19-9值等相关指标。ELISA检测患者血清中VEGF-C蛋白含量,并选择10例健康者血清作为对照。应用Kaplan-Meier法计算生存率,并绘制生存曲线,Log-rank法检验不同变量间生存率的差异,Cox回归模型进行预后的单因素分析和多因素分析。结果:35例胰腺癌患者血清中VEGF-C蛋白含量平均值为(1309.23±542.05)pg/ml,10例对照者为(256.13±96.59)pg/ml,差异有统计学意义(P<0.05)。将35例胰腺癌患者的年龄、肿瘤最大直径、肿瘤生长部位、KPS评分、血清CA19-9值及VEGF-C值等指标一起纳入Cox风险模型,进行预后单因素分析,发现KPS评分、血清CA19-9值及VEGF-C值均入选为无法切除的晚期胰腺癌患者预后影响因素(χ2=7.208、6.908、3.867,P=0.007、0.029、0.049)。对上述筛选变量进行多因素Cox回归模型分析,引入标准为0.10、删除标准为0.11,采用后退法,发现最先退出模型的是CA19-9值(χ2=3.738,P=0.053),然后是VEGF-C(χ2=4.873,P=0.027),最后是KPS评分(χ2=5.274,P=0.022),。比较KPS评分<70分与≥70分的两组病例预后,结果显示2组平均生存时间分别为6.60月与10.11月,1年累积生存率分别为21.43%与33.33%;取胰腺癌患者血清中VEGF-C蛋白含量中位数1280pg/ml为分界点,比较血清VEGF-C值≤1280pg/ml与>1280pg/ml的两组病例预后,结果显示2组平均生存时间分别为11.26月与6.29月,1年累积生存率分别为50.00%与5.88%;KPS评分及血清VEGF-C值的组间差异均有统计学意义(χ2=4.0400、9.4000,P=0.0443、0.0022)。而血清CA19-9值≤200U /ml与>200U/ml的两组病例,平均生存时间分别为9.99月与7.76月,1年累积生存率分别为37.50%与21.05%,差异无统计学意义(χ2=1.9100,P=0.1667)。结论:无法切除的局部晚期胰腺癌患者,其KPS评分和预后显著相关,可作为预后的独立影响因素;而血清中VEGF-C蛋白含量值与CA19-9值均和预后存在一定关系,可作为预后判断的辅助参考指标。更多还原

【Abstract】 PartⅠThe different expression of VEGF-C on primary tumor and lymph node metastasis of nude mice with orthotopic implantation for the human pancreatic cancer cellObjective:To investigate the different expression of vascular endothelial growth factor C (VEGF-C) on pancreatic cancer cell PANC-1 derived from primary tumor and spontaneous lymph node metastasis.Methods:Establishment of the spontaneous lymph node metastasis model in nude mice with orthotopic implantation for the human pancreatic cancer cell line PANC-1. Then, isolate and culture the pancreatic cancer cell from primary tumor and spontaneous lymph node metastasis. The different expression of VEGF-C on pancreatic cancer cell was detected by RT-PCR, Western Blot, ELISA.Results: The rate of tumor take was 100.0%. The rate of the spontaneous lymph node metastasis was 62.5%. The rate of the spontaneous hepatic metastasis was 25.0%. Not to see lung metastasis.The pancreatic cancer cell PANC-1 isolated from primary tumor and spontaneous lymph node metastasis were designated PANC-1-PRO and PANC-1-LN, separately. The mRNA levels of VEGF-C were 0.61±0.15 and 0.87±0.11 respectively; The protein levels of VEGF-C were 0.65±0.17 and 0.88±0.09 respectively inside 2 cells; Then the VEGF-C levels in culture supernatants were (1403.67±128.15) pg/ml and (1682.37±156.73) pg/ml. The levels of VEGF-C in PANC-1-LN cell were higher than PANC-1-PRO cell, the differentiation were statistical significance (t=3.1255、2.9062、3.0782,P=0.0141、0.0197、0.0152).Conclusions:The expressive level of VEGF-C on pancreatic cancer cell PANC-1 derived from spontaneous lymph node metastasis was higher than cell derived from primary tumor, the expression of VEGF-C on different organ was discrepancy.PartⅡEffect on apoptosis of pancreatic cancer derived from lymph node metastasis with antisense oligonucleotide of VEGF-CObjective:To investigate the effect of VEGF-C antisense oligonucleotide on pancreatic cancer cell PANC-1 derived from primary tumor and spontaneous lymph node metastasis.Methods: Establishment of the spontaneous lymph node metastasis model in nude mice with orthotopic implantation for the human pancreatic cancer cell line PANC-1. Then, isolate and culture the pancreatic cancer cell from primary tumor and spontaneous lymph node metastasis (PANC-1-PRO and PANC-1-LN). The cells were randomized into 3 groups: Control group, scramble-sense oligonucleotide (SODN) group and antisense oligonucleotide (ASODN) group. The effect of transfection on the expressive VEGF-C and apoptosis of pancreatic cancer cells were detected by RT-PCR, Western Blot, ELISA, flow cytometer and TUNEL in vitro and vivo.Results:After treatment in vitro, the VEGF-C level of mRNA, protein and culture supernatants on ASODN group of PANC-1-PRO and PANC-1-LN were decreased than control group and SODN group (P<0.01). In 3 groups of PANC-1-PRO and PANC-1-LN, the mRNA levels of apoptosis related gene bcl-2 were 0.67±0.12, 0.69±0.14, 0.61±0.11 and 0.56±0.16, 0.59±0.18, 0.27±0.17 separately. Only ASODN group of PANC-1-LN was decreased significantly (t=3.0428、3.1659,P=0.0124、0.0101). The rates of early apoptosis were (3.51±1.38)%, (4.79±2.16)%, (5.33±2.18)% in PANC-1-PRO, the differentiation were not statistical significance (t=1.7279、0.4310,P=0.1147、0.6756); But the rates were (2.83±1.01)%, (4.98±2.05)%, (13.22±2.17)% in PANC-1-LN, ASODN group was elevated, the differentiation were statistical significance (t=10.6329、6.7613,P=0.0000、0.0000);After treatment in vivo, the serum VEGF-C level of ASODN group were decreased than control group and SODN group in model(P<0.05). The volume of tumor, rates and parameter of lymph node metastasis all were not statistical differentiation (P>0.05). The rates of apoptosis were (1.29±0.53)%, (1.98±0.77)%, (2.01±0.80)% in 3 groups of primary tumor, the differentiation were not statistical significance (t=2.1221、0.0764,P=0.0522、0.9402); But in groups of lymph node metastasis, the rates of apoptosis were (1.78±0.49)%, (1.96±0.68)%, (4.38±1.02)% respectively, the rates of ASODN group was elevated, the differentiation were statistical significance (t=6.4987、5.2301,P=0.0000、0.0001).Conclusions:To inhibit the expression of VEGF-C on pancreatic cancer derived from lymph node metastasis can decreased the level of bcl-2, and promoted it to apoptosis, but no effect on pancreatic cancer derived from primary tumor. It is suggested that the effect of VEGF-C also have organic differentiation. It is mechanism of lymph node metastasis in pancreatic cancer.PartⅢThe correlation between the serum concentration of VEGF-C and prognosis in unrecetable pancreatic cancerObjective:To investigate the correlation between the serum concentration of VEGF-C and prognosis in unrecetable pancreatic cancer.Methods:In this study, select 35 unrecetable local advanced pancreatic cancer cases in Pancreatic Surgical Center of Union Hospital, Wuhan, China, from Dec. 2004 to Aug. 2005. We review karnofsky performance scale index (KPS), diameter of tumor and serum concentration of CA19-9. And the serum concentration of VEGF-C detected by ELISA, 10 health cases as to control. Survival rate and curves were calculated and drown by Kaplan-Meier. Survival rates in different groups were processed with Log-rank. Univariance and multiplevariance analysis were used to screen by Cox congression.Results:The mean serum concentration of VEGF-C was (1309.23±542.05) pg/ml, control group was (256.13±96.59) pg/ml, the differentiation were statistical significance (P<0.05).The patient year, diameter of tumor, position of tumor, KPS, the serum concentration of VEGF-C and CA19-9were univariance analysed by Cox congression. KPS, the serum concentration of CA19-9 and VEGF-C were all correlated with prognosis in unrecetable pancreatic cancer (χ2=7.208、6.908、3.867,P=0.007、0.029、0.049).In multiplevariance analysis by Cox congression (backward), SLE was 0.10, SLS was 0.11, the sequence of markers stepwise backward to model: Step 1 was VEGF-C (χ2=3.738,P=0.053), step 2 was VEGF-C (χ2=4.873,P=0.027), step 3 was KPS (χ2=5.274,P=0.022).In groups of KPS <70 and≥70分, The median survival were 6.60 and 10.11 months, the survival rates of 1 year were 21.43% and 33.33% respectively. As the median of VEGF-C was 1280pg/ml, in groups of≤1280pg/ml and >1280pg/ml, the median survival were 11.26 and 6.29 months, the survival rates of 1 year were 50.00% and 5.88% respectively, the differentiation were statistical significance (χ2=4.0400、9.4000,P=0.0443、0.0022). But, in groups of CA19-9≤200U /ml and >200U/ml, the median survival were 9.99 and 7.76 months, the survival rates of 1 year were 37.50% and 21.05% respectively, the differentiation were not statistical significance (χ2=1.9100,P=0.1667). Conclusions:KPS of the patients were independent factor of prognosis in unrecetable pancreatic cancer. The serum concentration of VEGF-C and CA19-9 were yet correlated with prognosis, and can used as aid marker for judgement of prognosis.更多还原