P16～(INK4a)基因在人胰腺癌中的表达及其作用机制的研究
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摘要
背景与目的
胰腺癌早期症状不典型，临床确诊时大多已处于晚期，化疗和
手术治疗效果不佳，5年生存率低，促使人们进行免疫、基因等方
面的研究，以期综合治疗改善预后。P16蛋白通过竞争性抑制细胞
周期中关键性的调节因子──Cyclins－CDKs复合物，细胞停滞在G1
晚期。已证实胰腺癌中常有P16蛋白的缺失，p16~（INK4a）基因的失活可
能是胰腺癌细胞恶性增殖的重要因素。本实验的目的为研究人胰腺
癌p16~（INK4a）基因表达的变化规律，并将野生型p16~（INK4a）基因导入人胰
腺癌细胞株，观察体外生长环境中p16~（INK4a）基因对细胞增殖的影响，
探讨p16~（INK4a）在胰腺癌发生发展中的表达意义、失活机制及可能的作
用机理，为临床诊断和治疗提供理论依据和新思路。
方法
1．用免疫组化方法及 Northern打点杂交方法研究人胰腺原发肿瘤和
非肿瘤组织中 P16蛋白和p16mRNA表达情况。
2．用PCR、Northern打点杂交及免疫组化方法分别对人胰腺癌细胞
株的p16~（INK4a）DNA、mRNA和蛋白表达进行分析。
3．用可表达P16蛋白的逆转录病毒pL-p16-SN和电穿孔方法将
p16~（INK4a）基因导入人胰腺癌细胞，通过ELISA、MTT、流式细胞
仪检测p16~（INK4a）的表达以及对细胞增殖、细胞周期和凋亡的影响。
结果
1．正常胰腺组织、胰腺良性病变及胰腺癌组织的p16蛋白表达水平
呈明显下降趋势（P＜0.01），胰腺癌P16蛋白表达与胰腺癌组织学分
级、临床分期的关系无显著差异（P＞0.05）。同一组织P16mRNA的
Northern打点杂交结果与P16蛋白检测结果相比阳性率稍高。
2．PCR结果示人胰腺癌细胞株Patu8902、Patu8988、SW1990无p16
Exon I 基因的缺失；Patu8902存在p16 Exon II 基因的缺失，胰腺
癌细胞株 Patu8988、SW1990未见 p16 Exon II基因缺失。PCR-SSCP
I
pl6”“’基困在人胰腺癌中的表达及其作用机制的研究（摘 要）
结果显示pl6”＊“基因在人胰腺癌细胞株PatU 8902、Patll8988、
O SW1990均无点突变存在。N。ribem打点杂交结果证实：人胰腺癌
细胞株P成U8902无p16 RNRNA的表达，P咖8988有p16 RNRNA表达，
SW 990呈弱表达。PI6蛋白表达结果显示，人胰腺癌细胞株
P ss8902呈阴性，P tltl 8988呈强阳性，Sw1990呈弱阳性表达。
3．分别通过逆转录病毒和电穿孔法将 pl6’毗“基因导入 pl6’叫‘“基因
缺失的人胰腺癌细胞 PatU8902，ELISA示 Pain8902－pl6可表达n 6
蛋白。流式细胞仪检测结果提示PatU8902、SW1990细胞在转染
pl6’毗“基因后的均产生凋亡峰。pl6”＊‘基因转染后 Pain8902和
SW1990细胞S期细胞明显减少，而 GdG；和G厂M的细胞增多。
O 病毒转染前后 PatU8988的细胞周期无明显变化。Hela细胞和
PW8988细胞的增殖不受P16病毒的影响（P＞o．OS）。PW89OZ和
SW1990细胞转染P16病毒后，细胞增殖明显受抑制，对SW1990
细胞增殖的影响尤为显著，Patll8902八 6细胞的增殖速度极为缓慢。
结论
1．pl6眺‘“基因失活与胰腺癌密切相关，在胰腺癌发生发展中可能主
要作用于肿瘤从良性向恶性转变的启动过程。
2．三株人胰腺癌细胞株中 pl6——“基因在 DNA、RNA和蛋白表达水
平上的检测结果表明 pl6”＊“基因纯合缺失是失活的重要机制。
3．pl6 CpG区的部分过甲基化也是造成 PI6蛋白缺失的重要机制之
一。另外，胰腺原发肿瘤在 pl6 InRNA向蛋白翻译过程中可能存
在其它抑制因素。
4．成功地将 pl6”＊‘基因导入 pl6”M‘基因缺失的人胰腺癌细胞
O Pain8902中，重新表达的 PI6蛋白使细胞失去恶性生长特性。
5．无或低水平表达 PI6蛋白的胰腺癌细胞在给予外源性 pl6”＊‘基固
后，可显著抑制癌细胞的增殖，促进癌细胞的凋亡，逆转癌细胞
的失控性增殖，提示pl6”＊“基固改变可能是胰腺癌癌变的重要机
制之一，同时亦预示了用 pl6’洲‘“基困对胰腺癌进行基固治疗具有
光明的前景，值得进一步探索。
Pancreatic carcinoma related symptoms occur late in disease
progression. Consequently, surgical intervention remains palliative in the
majority of patients. The prognoses of pancreatic cancer patients are
miserable even after radical surgery, and adjuvant therapy is necessary to
improve the surgical results. P16, encoded by p 1 6INK4a gene, is tight-
binding and inhibitory protein for cyclin-dependent kinase 4 to induce G 1
arrest of the cell cycle. p16 INK4a gene deletion is frequently identified in
human pancreas cancer. The impaired gene function of p16 might be a
major factor of the uncontrolled proliferation and malignancy of pancreas
cancer cells.
In this study, first, we studied human primary tumor tissues
neighboring normal tissues and cell lines for the alterations f 16 LNK4a
so as to clarigy the role of P16 protein in pancreatic adenocarcinoma.
Second, we investigated the effect of retroviral p16 expression vector for
pancreas cancer cell proliferation to clarify whether the vector might be a
promising mode to assist the surgical therapy for pancreas cancer.
By immunohistochemistry, We found the expression level of P16 of
pancreatic adenocarcinoma was much lower than that of benign disease
and normal tissues (P<0.0 1), but there was no differences in that of
different histopathologic grade and clinical stage. Northern blot showed
some tissues without P16 protein expression expressed p16 INK4a mRNA.
We studied 3 human pancreatic cell carcinoma lines, and found that
Patu8902 revealed homozygous deletion of ~16 INK4a exon II and SW 1990
had a quite low expression of P16 protein. Then We used the retroviral
p16 expression vector pL-p 16-SN, which inserting p16 cDNA to a vector
containing w + transcription and processing elements replication-
incompetent infectious virus, to transfect pancreas cancer cell lines.
Thereafter, we assessed the activity of pL-p 16-SN to induce p16 gene
mRNA expression in Patu8902-p16 and to control cell proliferation. By
3
nowcytometric stUdies, Patu89o2 and swl99o cell was arrested at G,-G,
and Gz-M stage after pL-p16-SN trallsfection. The cell proliferation was
significanly suPPressed by pL-pl6-SN compared with the control grouP
(P<0.0l). These data indicate that the dysfunction of pl6 INK4a gene in
pancreatic carcinoma is frequently, and pL-pl6-SN has the potential to
induce pl6 gene expression and control pancreas cancer cell proliferation.
And it suggest that the retroviral pl6 expression vector pL-pl6-SN might
be a possible method of gene theraPy to improve the surgical theraPeutic
results for pancreas cancer.
胰腺癌早期症状不典型，临床确诊时大多已处于晚期，化疗和
手术治疗效果不佳，5年生存率低，促使人们进行免疫、基因等方
面的研究，以期综合治疗改善预后。P16蛋白通过竞争性抑制细胞
周期中关键性的调节因子──Cyclins－CDKs复合物，细胞停滞在G1
晚期。已证实胰腺癌中常有P16蛋白的缺失，p16~（INK4a）基因的失活可
能是胰腺癌细胞恶性增殖的重要因素。本实验的目的为研究人胰腺
癌p16~（INK4a）基因表达的变化规律，并将野生型p16~（INK4a）基因导入人胰
腺癌细胞株，观察体外生长环境中p16~（INK4a）基因对细胞增殖的影响，
探讨p16~（INK4a）在胰腺癌发生发展中的表达意义、失活机制及可能的作
用机理，为临床诊断和治疗提供理论依据和新思路。
方法
1．用免疫组化方法及 Northern打点杂交方法研究人胰腺原发肿瘤和
非肿瘤组织中 P16蛋白和p16mRNA表达情况。
2．用PCR、Northern打点杂交及免疫组化方法分别对人胰腺癌细胞
株的p16~（INK4a）DNA、mRNA和蛋白表达进行分析。
3．用可表达P16蛋白的逆转录病毒pL-p16-SN和电穿孔方法将
p16~（INK4a）基因导入人胰腺癌细胞，通过ELISA、MTT、流式细胞
仪检测p16~（INK4a）的表达以及对细胞增殖、细胞周期和凋亡的影响。
结果
1．正常胰腺组织、胰腺良性病变及胰腺癌组织的p16蛋白表达水平
呈明显下降趋势（P＜0.01），胰腺癌P16蛋白表达与胰腺癌组织学分
级、临床分期的关系无显著差异（P＞0.05）。同一组织P16mRNA的
Northern打点杂交结果与P16蛋白检测结果相比阳性率稍高。
2．PCR结果示人胰腺癌细胞株Patu8902、Patu8988、SW1990无p16
Exon I 基因的缺失；Patu8902存在p16 Exon II 基因的缺失，胰腺
癌细胞株 Patu8988、SW1990未见 p16 Exon II基因缺失。PCR-SSCP
I
pl6”“’基困在人胰腺癌中的表达及其作用机制的研究（摘 要）
结果显示pl6”＊“基因在人胰腺癌细胞株PatU 8902、Patll8988、
O SW1990均无点突变存在。N。ribem打点杂交结果证实：人胰腺癌
细胞株P成U8902无p16 RNRNA的表达，P咖8988有p16 RNRNA表达，
SW 990呈弱表达。PI6蛋白表达结果显示，人胰腺癌细胞株
P ss8902呈阴性，P tltl 8988呈强阳性，Sw1990呈弱阳性表达。
3．分别通过逆转录病毒和电穿孔法将 pl6’毗“基因导入 pl6’叫‘“基因
缺失的人胰腺癌细胞 PatU8902，ELISA示 Pain8902－pl6可表达n 6
蛋白。流式细胞仪检测结果提示PatU8902、SW1990细胞在转染
pl6’毗“基因后的均产生凋亡峰。pl6”＊‘基因转染后 Pain8902和
SW1990细胞S期细胞明显减少，而 GdG；和G厂M的细胞增多。
O 病毒转染前后 PatU8988的细胞周期无明显变化。Hela细胞和
PW8988细胞的增殖不受P16病毒的影响（P＞o．OS）。PW89OZ和
SW1990细胞转染P16病毒后，细胞增殖明显受抑制，对SW1990
细胞增殖的影响尤为显著，Patll8902八 6细胞的增殖速度极为缓慢。
结论
1．pl6眺‘“基因失活与胰腺癌密切相关，在胰腺癌发生发展中可能主
要作用于肿瘤从良性向恶性转变的启动过程。
2．三株人胰腺癌细胞株中 pl6——“基因在 DNA、RNA和蛋白表达水
平上的检测结果表明 pl6”＊“基因纯合缺失是失活的重要机制。
3．pl6 CpG区的部分过甲基化也是造成 PI6蛋白缺失的重要机制之
一。另外，胰腺原发肿瘤在 pl6 InRNA向蛋白翻译过程中可能存
在其它抑制因素。
4．成功地将 pl6”＊‘基因导入 pl6”M‘基因缺失的人胰腺癌细胞
O Pain8902中，重新表达的 PI6蛋白使细胞失去恶性生长特性。
5．无或低水平表达 PI6蛋白的胰腺癌细胞在给予外源性 pl6”＊‘基固
后，可显著抑制癌细胞的增殖，促进癌细胞的凋亡，逆转癌细胞
的失控性增殖，提示pl6”＊“基固改变可能是胰腺癌癌变的重要机
制之一，同时亦预示了用 pl6’洲‘“基困对胰腺癌进行基固治疗具有
光明的前景，值得进一步探索。
Pancreatic carcinoma related symptoms occur late in disease
progression. Consequently, surgical intervention remains palliative in the
majority of patients. The prognoses of pancreatic cancer patients are
miserable even after radical surgery, and adjuvant therapy is necessary to
improve the surgical results. P16, encoded by p 1 6INK4a gene, is tight-
binding and inhibitory protein for cyclin-dependent kinase 4 to induce G 1
arrest of the cell cycle. p16 INK4a gene deletion is frequently identified in
human pancreas cancer. The impaired gene function of p16 might be a
major factor of the uncontrolled proliferation and malignancy of pancreas
cancer cells.
In this study, first, we studied human primary tumor tissues
neighboring normal tissues and cell lines for the alterations f 16 LNK4a
so as to clarigy the role of P16 protein in pancreatic adenocarcinoma.
Second, we investigated the effect of retroviral p16 expression vector for
pancreas cancer cell proliferation to clarify whether the vector might be a
promising mode to assist the surgical therapy for pancreas cancer.
By immunohistochemistry, We found the expression level of P16 of
pancreatic adenocarcinoma was much lower than that of benign disease
and normal tissues (P<0.0 1), but there was no differences in that of
different histopathologic grade and clinical stage. Northern blot showed
some tissues without P16 protein expression expressed p16 INK4a mRNA.
We studied 3 human pancreatic cell carcinoma lines, and found that
Patu8902 revealed homozygous deletion of ~16 INK4a exon II and SW 1990
had a quite low expression of P16 protein. Then We used the retroviral
p16 expression vector pL-p 16-SN, which inserting p16 cDNA to a vector
containing w + transcription and processing elements replication-
incompetent infectious virus, to transfect pancreas cancer cell lines.
Thereafter, we assessed the activity of pL-p 16-SN to induce p16 gene
mRNA expression in Patu8902-p16 and to control cell proliferation. By
3
nowcytometric stUdies, Patu89o2 and swl99o cell was arrested at G,-G,
and Gz-M stage after pL-p16-SN trallsfection. The cell proliferation was
significanly suPPressed by pL-pl6-SN compared with the control grouP
(P<0.0l). These data indicate that the dysfunction of pl6 INK4a gene in
pancreatic carcinoma is frequently, and pL-pl6-SN has the potential to
induce pl6 gene expression and control pancreas cancer cell proliferation.
And it suggest that the retroviral pl6 expression vector pL-pl6-SN might
be a possible method of gene theraPy to improve the surgical theraPeutic
results for pancreas cancer.
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53． Coffin JM & Varmus HE. Retroviruses. Cold Spring Harbor Laboratory Press, NY. 1996
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