抗人肝肠钙黏附蛋白（CDH17）单克隆抗体治疗肝癌的实验研究
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摘要
第一部分
抗人肝肠钙黏附蛋白(CDH17)单克隆抗体的制备、纯化及鉴定
目的：
培育Lic5及Lic3杂交瘤细胞,通过小鼠腹腔接种杂交瘤细胞的方法制备纯化抗人肝肠钙黏附蛋白(CDH17)单克隆抗体Lic5及Lic3,用以研究以人CDH17为靶点的单克隆抗体的体内外抗肿瘤作用。
方法：
将Lic5及Lic3杂交瘤细胞注射入BALB/c nu/nu小鼠腹腔,收集含有单克隆抗体的腹水。使用Protein A亲和层析法纯化单克隆抗体,Western Blot鉴定单克隆抗体的特异性,银染色法鉴定单克隆抗体的纯度,改良Lowry法测定抗体浓度。
结果：
1.Lic5及Lic3杂交瘤细胞生长良好,活力高。裸鼠腹腔注射Lic5杂交瘤7-10天即可收集腹水,平均产量7.25ml/只裸鼠,注射Lic3杂交瘤第15天可收集腹水,平均产量5.5ml／只裸鼠。
2.Lic5及Lic3银染色在50kD和25kD处各见清晰独立条带,分别为单克隆抗体的重链和轻链位置,无明显杂带。Western Blot测定MHCC97L及AGS细胞裂解液CDH17表达,在120kD处可见到单一条带,无明显杂带。通过亚型试纸鉴定,Lic5及Lic3单克隆抗体分别为IgG2b、IgG2a。
3.Lic5杂交瘤腹水中单克隆抗体含量为2.75mg/ml, Lic3杂交瘤腹水中单克隆抗体含量11.8mg/ml。
结论
1.通过杂交瘤腹水种植法获得高产量、高效价的抗CDH17单克隆抗体腹水。
2.成功制备了高产量、高纯度、高特异度的抗人CDH17单克隆抗体Lic5及Lic3。
3. Protein A亲和层析法适合于实验室用单克隆抗体的纯化。
第二部分
抗人肝肠钙黏附蛋白(CDH17)单克隆抗体对人肝癌细胞株的生物学属性的影响
目的：
研究抗CDH17单克隆抗体Lic5及Lic3对CDH17不同表达水平的人肝癌细胞株MHCC97L, MHCC97H, PLC/PRF/5及永生型人肝细胞株MIHA的增殖、迁移、侵袭、克隆形成及对针对性化疗药物敏感性的变化,探讨其作用机制。
方法：
用Western Blot及qPCR方法检测四种细胞株MHCC97L, MHCC97H, PLC/PRF/5及MIHA的CDH17表达水平；细胞免疫荧光法检测单克隆抗体Lic5及Lic3与活细胞膜上的CDH17的结合能力。以PBS和小鼠IgG为对照,MTT法检测细胞的增殖能力；细胞划痕实验检测细胞的迁移能力；Transwell法检测细胞的侵袭能力；平板法检测细胞的克隆形成能力；MTT法检测细胞对化疗药物顺铂的敏感性变化；Western Blot进行相关信号通路的初步探讨。
结果：
1.CDH17在人肝癌转移细胞株MHCC97L、MHCC97H中明显高表达,而在人肝癌原位细胞株PLC/PRF/5及永生型人肝细胞株MIHA中检测不到蛋白水平表达,mRNA水平明显低表达。
2.免疫荧光染色显示MHCC97L及MHCC97H细胞在单克隆抗体Lic5及Lic3组可以见到细胞膜强荧光染色,而在PLC/PRF/5和MIHA细胞株未见细胞膜荧光染色。
3. MHCC97L及MHCC97H细胞的增殖能力在单克隆抗体Lic5及Lic3组明显减弱,与对照组比较有统计学意义(p<0.05),而PLC/PRF/5及MIHA细胞增殖能力在各组无差异。
4. MHCC97L及MHCC97H细胞的迁移能力在单克隆抗体Lic5及Lic3组明显减弱,与对照组比较有统计学意义(p<0.05),而PLC/PRF/5及MIHA细胞在各组迁移能力无差异。
5. MHCC97L及MHCC97H细胞的穿膜细胞数在单克隆抗体Lic5及Lic3组明显减少,MHCC97L穿膜细胞抑制率分别是78.9%和71.1%,MHCC97H穿膜细胞抑制率分别是67.3%和60.9%,与对照组比较有统计学意义(p<0.01),而PLC/PRF/5及MIHA细胞穿膜细胞数在各组无明显差异。
6. MHCC97L及MHCC97H细胞的平板克隆形成数在单克隆抗体Lic5及Lic3组明显减少,MHCC97L平板克隆形成抑制率分别是53%和48.6%,MHCC97H平板克隆抑制率分别是60.5%和56.3%,与对照组比较有统计学意义(p<0.01),而PLC/PRF/5及MIHA细胞平板克隆细胞数在各组均无明显差异。
7.MHCC97L及MHCC97H细胞对顺铂(Cisplatin)的敏感性检测,单克隆抗体Lic5及Lic3组细胞存活率与对照组相比,差异有统计学意义(p<0.05),而对PLC/PRF/5及MIHA细胞敏感性无明显影响。
8.Lic5及Lic3单克隆抗体可以下调MHCC97L及MHCC97H细胞的CDH17蛋白表达,对mRNA表达水平无影响。
9. MHCC97L及MHCC97H细胞中,与对照组相比,Wnt/β-catenin信号通路关键分子β-catenin在Lic5及Lic3单克隆抗体组表达水平明显降低。
结论：
1.Lic5及Lic3单克隆抗体可以和MHCC97L及MHCC97H活细胞细胞膜上的CDH17结合。
2.与PBS空白对照组及小鼠IgG平行对照组相比,Lic5及Lic3单克隆抗体明显改变MHCC97L及MHCC97H细胞生物学属性,表现为增殖迟缓,迁移、侵袭能力减退,平板克隆能力减弱。
3.Lic5及Lic3单克隆抗体明显提高MHCC97L及MHCC97H细胞对顺铂的敏感性。
4.Lic5及Lic3单克隆抗体可以下调MHCC97L及MHCC97H细胞的CDH17及β-catenin蛋白表达。
第三部分
抗人肝肠钙黏附蛋白(CDH17)单克隆抗体及联合顺铂(Cisplatin)靶向治疗对活体肝癌生长、转移的抑制作用及相关信号通路的初步探讨
目的：
研究抗人CDH17单克隆抗体Lic5及Lic3及联合顺铂(Cisplatin)对人肝癌细胞MHCC97L皮下移植瘤及原位移植瘤的抗肿瘤作用,探讨相关信号通路的改变。
方法：
用人肝癌细胞株MHCC97L分别建立皮下移植瘤模型和原位移植瘤模型。将皮下移植瘤模型裸鼠分为六组,分别是PBS空白对照组,小鼠IgG平行对照组,Lic5低剂量组(2.5mg/kg), Lic5高剂量组(5mg/kg),顺铂单药治疗组(lmg/kg),Lic5联合顺铂治疗组(Lic55mg/kg,顺铂lmg/kg),观察Lic5单克隆抗体及联合顺铂对皮下移植瘤生长的影响。用Western Blot及免疫组织化学方法检测人肝癌皮下荷瘤鼠单克隆抗体治疗后肿瘤组织内CDH17表达变化及相关信号通路关键分子的表达变化。将原位移植瘤模型裸鼠分为四组,PBS空白对照组,小鼠IgG平行对照组,Lic5低剂量组(2.5mg/kg),Lic5高剂量组(5mg/kg),使用生物发光监测不同处理组肿瘤生长的变化。
结果：
1.在MHCC97L皮下移植瘤模型中,与PBS空白对照组及小鼠IgG平行对照组比较,Lic5高低剂量组及联合顺铂治疗组皮下瘤均生长缓慢,以Lic5联合顺铂治疗组肿瘤生长最慢,差异具有统计学意义((p<0.05)。Lic5联合顺铂组肿瘤生长较顺铂单用组明显变慢,差异具有统计学意义(p<0.01)。
2.在MHCC97L皮下移植瘤模型中,Lic5低剂量组(2.5mg/kg)肺转移率50%,高剂量组(5mg/kg) 33%,Lic5与顺铂联合组0%,而PBS空白对照组及小鼠IgG平对照组均为83.3%。
3.在MHCC97L皮下移植瘤模型中,Lic5高低剂量组及联合顺铂治疗组肿瘤组织内Ki67阳性细胞数目明显减少,与PBS空白对照组及小鼠IgG平行对照组比较,差异具有统计学意义(p<0.001)。
4.在MHCC97L皮下移植瘤模型中,Western Blot及免疫组化染色均显示Lic5高低剂量组及联合顺铂治疗组肿瘤组织中CDH17表达减少,以Lic5高剂量组及联合顺铂组显著。在CDH17下调同时,也检测到Wnt/β-catenin信号通路中关键分子β-catenin、Cyclin D1表达均减少,而Rb表达相应增加。
5.在MHCC97L皮下移植瘤模型中,H&E染色显示Lic5高低剂量组及联合顺铂治疗组裸鼠肝、肾、肺、脾等重要器官未发现明显组织结构异常。
6.在MHCC97L原位移植瘤模型中,与PBS空白对照组及小鼠IgG平行对照组比较,Lic5高低剂量治疗组生物发光信号均减弱,原位肿瘤体积明显小于对照组,差异具有统计学意义(p<0.05)。在PBS组及小鼠IgG对照组,肺转移率100%,在Lic5低剂量组(2.5mg/kg),肺转移率50%,在Lic5高剂量组(5mg/kg),肺转移率33.3%。结果与MHCC97L皮下移植瘤治疗结果一致。
结论：
1.抗人CDH17单克隆抗体Lic5能够显著抑制MHCC97L裸鼠皮下移植瘤及原位移植瘤的生长和肺转移,为肝癌的分子靶向治疗提供了有效的实验依据。
2.抗人CDH17单克隆抗体Lic5通过下调MHCC97L裸鼠皮下瘤组织中CDH17表达水平,失活Wnt/β-Catenin信号通路发挥抗肿瘤作用。
3.抗人CDH17单克隆抗体Lic5可以增加MHCC97L裸鼠皮下瘤细胞对顺铂的敏感性,为肿瘤的多药联合治疗提供了有效的实验依据。
4.抗人CDH17单克隆抗体Lic5在活体治疗中未发现明显生物学安全性隐患。
PartI
Preparation, purification and identification of monoclonal antibodies against human cadherin 17(CDH17)
Purpose:
Prepare Lic5 and Lic3 monoclonal antibodies (mAbs) from nude mouse ascites induced by Lic5 and Lic3 hybridoma cells and identify the purity and specificity of mAbs for the study of monoclonal antibody therapy in human hepatocellular carcinoma (HCC).
Materials and Methods:
Lic5 and Lic3 hybridoma cells were injected into abdominal cavity of BALB/c nu/nu mice and collected the ascites containing the mAbs. Purify Lic5 and Lic3 mAbs from mouse ascites by Protein A affinity chromatography and identify mAbs'purity and specificity by Western Blot and silver staining. Measure mAbs'concentration by Lowry method.
Results:
1. Lic5 and Lic3 hybridoma cells grew well in high activity. Ascites was collected 7-10 days after Lic5 hybridoma cell injection and 15 days after Lic3 hybridoma cell injection. The average yield of ascites with Lic5 and Lic3 hybridoma cells were 7.25ml and 5.5ml per nude mouse respectively.
2. From the silver staining of Lic5 and Lic3 mAbs, we only found two pieces of clear and independent bands at 50kD and 25kD, which represent the mAbs' heavy chain and light chain respectively. Western Blot detected CDH17 in cell lysate of MHCC97L and AGS with a single band at 120kD. Isotypes of Lic5 and Lic3 were identified by test paper and mAbs were IgG2b and IgG2a respectively.
3. Lic5 and Lic3 mAbs output of hybridoma ascites were 2.75mg/ml and 11.8mg/ml respectively.
Conclusion:
1. Mouse ascites containing Lic5 and Lic3 mAbs were harvested with high quality and high titer.
2. Lic5 and Lic3 mAbs against human CDH17 were prepared successfully with high purity and specificity.
3. Protein A affinity chromatography is suitable for purification of mAbs in the laboratory.
Part II
Characterization of changes in biological properties of human hepatocellualr carcinoma cell lines under the anti-CDH17 mAbs
Purpose:
To study the changes in cell proliferation, migration, invasion, colony formation and related drug sensitivity of human hepatoma cell lines MHCC97L, MHCC97H, PLC/PRF/5 and immortalized human hepatocyte MIHA under the Lic5 and Lic3 mAbs'treatment. To explore the possible underlying mechanisms.
Materials and Methods:
Detect the CDH17 protein and mRNA level in four cell lines such as MHCC97L, MHCC97H, PLC/PRF/5 and MIHA by the way of Western Blot and quantitative real-time PCR. Examine the binding capacity between Lic5 and Lic3 mAbs and CDH17 in the living cell membrane by the way of immunofluorescence staining. With the PBS and mouse IgG as the control, cell proliferation was detected by the way of MTT assay, cell migration was checked by wound healing assay, cell invasion assay was calculated by transwell method, cell colony formation was detected by plate colony assay. Drug sensitivity assay was monitored by MTT assay. Signaling pathways was explored by Western Blot.
Results:
1. CDH17 protein and mRNA were detected with a strongly high level in metastatic HCC cell lines MHCC97L, MHCC97H, but very little or undetectable level in primary HCC cell line PLC/PRF/5 and immortalized normal human hepatocyte cell line MIHA.
2. Lic5 and Lic3 mAbs can bind with the CDH17 in the membrane of living cell lines MHCC9L and MHCC97H.
3. Cell proliferation ability of MHCC97L and MHCC97H in mAbs'groups was significantly weakened. The statistic analysis showed significant difference between mAbs'groups and controls (p<0.05). There was no difference between mAbs'groups and controls in PLC/PRF/5, and MIHA cell lines.
4. The number of clone formation, migration and invasion in Lic5 and Lic3 mAbs' groups of MHCC97L and MHCC97H cells was significantly less than that in PBS and mouse Ig groups. Statistic analysis showed significant difference between mAbs'and control groups.
5. The drug sensitivity and inhibition rate of cisplatin to MHCC97L and MHCC97H cells was significantly elevated as compared to PBS and mouse Ig controls.
6. Lic5 and Lic3 mAbs can downregulate the CDH17 protein expression level in MHCC97L and MHCC97H cells and had no any effect on CDH17 mRNA level.
7. Lic5 and Lic3 mAbs can downregulate theβ-catenin protein expression level in MHCC97L and MHCC97H cells, which was the key molecule in Wnt/p-catenin signaling pathway.
Conclusion:
1. Lic5 and Lic3 mAbs can bind with CDH17 in cell membrane in living MHCC97L and MHCC97H cells.
2. The MHCC97L and MHCC97H cells in mAs'groups showed great changes in the cell biological properties as compared to PBS and mouse Ig controls including retarded proliferation; decreased ability in cell clone formation, cell migration and invasion.
3. Lic5 and Lic3 mAbs increased the drug sensititvity of MHCC97L and MHCC97H to cisplatin significantly.
4. The CDH17 andβ-catenin protein level were significantly reduced by Lic5 and Lic3 mAbs in MHCC97L and MHCC97H cells.
Part III
The inhibition role of anti-CDH17 monoclonal antibody and combination with cisplatin on hepatocellular carcinoma in vivo and the exploration of the anti-tumor mechanisms.
Purpose:
To studay the anti-tumor effect of Lic5 and the combination between Lic5 and cisplatin in subcutaneous xenograft model and orthotopic transplantation model of MHCC97L cell. To explore the underlying signaling pathway related to the mAbs' therapy.
Materials and Methods:
Use MHCC97L or with luciferase transfection to establish the subcutaneous tumor model and orthotopic tansplantation tumor model of human HCC in nude mouse. The subcutaneous tumor models in nude mice were divided into six groups, PBS empty control group, Mouse IgG parallel control group, Lic5 mAb low dosage group(2.5mg/kg), Lic5 mAb high dosage group(5mg/kg), cisplatin group(lmg/kg), the combination group of Lic5 mAb and cisplatin(Lic55mg/kg, cisplatin lmg/kg). Observe the impact of Lic5 and the combination with cisplatin on subcutaneous tumor growth. Detect the change of expression level of CDH17 and key molecules in related signaling pathway by the way of Western Blot. The orthotopic transplantation tumor models in nude mice were divided into four groups, PBS empty control group, Mouse IgG parallel control group, Lic5 mAb low dosage group(2.5mg/kg), Lic5 mAb high dosage group(5mg/kg). Use the bioluminescence to montitor the tumr growth in different groups.
Results:
1. Compared with PBS and mouse Ig control in subcutaneous model, the tumor growth was remarkably inhibited in mAbs'groups. Among the former three groups, the tumor growth in Lic5 and cisplatin combination group showed the strongest inhibition. Statistics analysis showed significant difference between the mAbs'and control gourps.
2. In the subcutaneous tumor models, the lung metastasis rate in PBS and mouse Ig groups were all 83.3%, Lic5 low dosage, high dosage and combination groups were 50%,33%and 0.
3. The number of Ki67 positive cells were significantly decreased in mAbs'groups compared with PBS and mouse Ig groups in subcutaneous tumor models. Statistics analysis showed significant difference between mAbs'and control groups.
4. Western Blot showed the expression of CDH17 was significantly downregulated in mAbs'treatment groups whereas high expression of CDH17 in control groups. The key molecules in Wnt/β-catenin, (3-catenin and Cyclin D1 were also downregulated in mAbs'treatment groups, but Rb was upregulated in the same groups.
5. There was no obvious tissue abnormalities in liver, kidney, lung and spleen organs in mAbs'treatment groups.
6. Compared with the PBS and moue Ig controls in orthotopic tumor models, bioluminescence signals and tumor volume were reduced significantly in mAs' treatment groups. Statistics analysis showed significant difference between mAbs' groups and control groups. The tumor lung metastasis rate in PBS and mouse Ig groups were all 100%and 50%,33.3%in Lic5 low and high dosage groups.
Conclusion:
1. Anti-CDH17 monoclonal antibody Lic5 can significantly inhibit the MHCC97L tumor growth and lung metastasis in nude mouse model and is a promising candidate in HCC targeted therapy.
2. Lic5 mAb inhibited MHCC97L tumor growth by downregulation of CDH17 and inactivation of Wnt/β-catenin signaling pathway.
3. Lic5 mAb can improve the drug sensitivity to cisplatin in MHCC97L in vivo and provide an effective experimental basis for the multi-drug combination therapy in HCC.
4. No risk in biological safety had been observed during the period of mAb administration.
抗人肝肠钙黏附蛋白(CDH17)单克隆抗体的制备、纯化及鉴定
目的：
培育Lic5及Lic3杂交瘤细胞,通过小鼠腹腔接种杂交瘤细胞的方法制备纯化抗人肝肠钙黏附蛋白(CDH17)单克隆抗体Lic5及Lic3,用以研究以人CDH17为靶点的单克隆抗体的体内外抗肿瘤作用。
方法：
将Lic5及Lic3杂交瘤细胞注射入BALB/c nu/nu小鼠腹腔,收集含有单克隆抗体的腹水。使用Protein A亲和层析法纯化单克隆抗体,Western Blot鉴定单克隆抗体的特异性,银染色法鉴定单克隆抗体的纯度,改良Lowry法测定抗体浓度。
结果：
1.Lic5及Lic3杂交瘤细胞生长良好,活力高。裸鼠腹腔注射Lic5杂交瘤7-10天即可收集腹水,平均产量7.25ml/只裸鼠,注射Lic3杂交瘤第15天可收集腹水,平均产量5.5ml／只裸鼠。
2.Lic5及Lic3银染色在50kD和25kD处各见清晰独立条带,分别为单克隆抗体的重链和轻链位置,无明显杂带。Western Blot测定MHCC97L及AGS细胞裂解液CDH17表达,在120kD处可见到单一条带,无明显杂带。通过亚型试纸鉴定,Lic5及Lic3单克隆抗体分别为IgG2b、IgG2a。
3.Lic5杂交瘤腹水中单克隆抗体含量为2.75mg/ml, Lic3杂交瘤腹水中单克隆抗体含量11.8mg/ml。
结论
1.通过杂交瘤腹水种植法获得高产量、高效价的抗CDH17单克隆抗体腹水。
2.成功制备了高产量、高纯度、高特异度的抗人CDH17单克隆抗体Lic5及Lic3。
3. Protein A亲和层析法适合于实验室用单克隆抗体的纯化。
第二部分
抗人肝肠钙黏附蛋白(CDH17)单克隆抗体对人肝癌细胞株的生物学属性的影响
目的：
研究抗CDH17单克隆抗体Lic5及Lic3对CDH17不同表达水平的人肝癌细胞株MHCC97L, MHCC97H, PLC/PRF/5及永生型人肝细胞株MIHA的增殖、迁移、侵袭、克隆形成及对针对性化疗药物敏感性的变化,探讨其作用机制。
方法：
用Western Blot及qPCR方法检测四种细胞株MHCC97L, MHCC97H, PLC/PRF/5及MIHA的CDH17表达水平；细胞免疫荧光法检测单克隆抗体Lic5及Lic3与活细胞膜上的CDH17的结合能力。以PBS和小鼠IgG为对照,MTT法检测细胞的增殖能力；细胞划痕实验检测细胞的迁移能力；Transwell法检测细胞的侵袭能力；平板法检测细胞的克隆形成能力；MTT法检测细胞对化疗药物顺铂的敏感性变化；Western Blot进行相关信号通路的初步探讨。
结果：
1.CDH17在人肝癌转移细胞株MHCC97L、MHCC97H中明显高表达,而在人肝癌原位细胞株PLC/PRF/5及永生型人肝细胞株MIHA中检测不到蛋白水平表达,mRNA水平明显低表达。
2.免疫荧光染色显示MHCC97L及MHCC97H细胞在单克隆抗体Lic5及Lic3组可以见到细胞膜强荧光染色,而在PLC/PRF/5和MIHA细胞株未见细胞膜荧光染色。
3. MHCC97L及MHCC97H细胞的增殖能力在单克隆抗体Lic5及Lic3组明显减弱,与对照组比较有统计学意义(p<0.05),而PLC/PRF/5及MIHA细胞增殖能力在各组无差异。
4. MHCC97L及MHCC97H细胞的迁移能力在单克隆抗体Lic5及Lic3组明显减弱,与对照组比较有统计学意义(p<0.05),而PLC/PRF/5及MIHA细胞在各组迁移能力无差异。
5. MHCC97L及MHCC97H细胞的穿膜细胞数在单克隆抗体Lic5及Lic3组明显减少,MHCC97L穿膜细胞抑制率分别是78.9%和71.1%,MHCC97H穿膜细胞抑制率分别是67.3%和60.9%,与对照组比较有统计学意义(p<0.01),而PLC/PRF/5及MIHA细胞穿膜细胞数在各组无明显差异。
6. MHCC97L及MHCC97H细胞的平板克隆形成数在单克隆抗体Lic5及Lic3组明显减少,MHCC97L平板克隆形成抑制率分别是53%和48.6%,MHCC97H平板克隆抑制率分别是60.5%和56.3%,与对照组比较有统计学意义(p<0.01),而PLC/PRF/5及MIHA细胞平板克隆细胞数在各组均无明显差异。
7.MHCC97L及MHCC97H细胞对顺铂(Cisplatin)的敏感性检测,单克隆抗体Lic5及Lic3组细胞存活率与对照组相比,差异有统计学意义(p<0.05),而对PLC/PRF/5及MIHA细胞敏感性无明显影响。
8.Lic5及Lic3单克隆抗体可以下调MHCC97L及MHCC97H细胞的CDH17蛋白表达,对mRNA表达水平无影响。
9. MHCC97L及MHCC97H细胞中,与对照组相比,Wnt/β-catenin信号通路关键分子β-catenin在Lic5及Lic3单克隆抗体组表达水平明显降低。
结论：
1.Lic5及Lic3单克隆抗体可以和MHCC97L及MHCC97H活细胞细胞膜上的CDH17结合。
2.与PBS空白对照组及小鼠IgG平行对照组相比,Lic5及Lic3单克隆抗体明显改变MHCC97L及MHCC97H细胞生物学属性,表现为增殖迟缓,迁移、侵袭能力减退,平板克隆能力减弱。
3.Lic5及Lic3单克隆抗体明显提高MHCC97L及MHCC97H细胞对顺铂的敏感性。
4.Lic5及Lic3单克隆抗体可以下调MHCC97L及MHCC97H细胞的CDH17及β-catenin蛋白表达。
第三部分
抗人肝肠钙黏附蛋白(CDH17)单克隆抗体及联合顺铂(Cisplatin)靶向治疗对活体肝癌生长、转移的抑制作用及相关信号通路的初步探讨
目的：
研究抗人CDH17单克隆抗体Lic5及Lic3及联合顺铂(Cisplatin)对人肝癌细胞MHCC97L皮下移植瘤及原位移植瘤的抗肿瘤作用,探讨相关信号通路的改变。
方法：
用人肝癌细胞株MHCC97L分别建立皮下移植瘤模型和原位移植瘤模型。将皮下移植瘤模型裸鼠分为六组,分别是PBS空白对照组,小鼠IgG平行对照组,Lic5低剂量组(2.5mg/kg), Lic5高剂量组(5mg/kg),顺铂单药治疗组(lmg/kg),Lic5联合顺铂治疗组(Lic55mg/kg,顺铂lmg/kg),观察Lic5单克隆抗体及联合顺铂对皮下移植瘤生长的影响。用Western Blot及免疫组织化学方法检测人肝癌皮下荷瘤鼠单克隆抗体治疗后肿瘤组织内CDH17表达变化及相关信号通路关键分子的表达变化。将原位移植瘤模型裸鼠分为四组,PBS空白对照组,小鼠IgG平行对照组,Lic5低剂量组(2.5mg/kg),Lic5高剂量组(5mg/kg),使用生物发光监测不同处理组肿瘤生长的变化。
结果：
1.在MHCC97L皮下移植瘤模型中,与PBS空白对照组及小鼠IgG平行对照组比较,Lic5高低剂量组及联合顺铂治疗组皮下瘤均生长缓慢,以Lic5联合顺铂治疗组肿瘤生长最慢,差异具有统计学意义((p<0.05)。Lic5联合顺铂组肿瘤生长较顺铂单用组明显变慢,差异具有统计学意义(p<0.01)。
2.在MHCC97L皮下移植瘤模型中,Lic5低剂量组(2.5mg/kg)肺转移率50%,高剂量组(5mg/kg) 33%,Lic5与顺铂联合组0%,而PBS空白对照组及小鼠IgG平对照组均为83.3%。
3.在MHCC97L皮下移植瘤模型中,Lic5高低剂量组及联合顺铂治疗组肿瘤组织内Ki67阳性细胞数目明显减少,与PBS空白对照组及小鼠IgG平行对照组比较,差异具有统计学意义(p<0.001)。
4.在MHCC97L皮下移植瘤模型中,Western Blot及免疫组化染色均显示Lic5高低剂量组及联合顺铂治疗组肿瘤组织中CDH17表达减少,以Lic5高剂量组及联合顺铂组显著。在CDH17下调同时,也检测到Wnt/β-catenin信号通路中关键分子β-catenin、Cyclin D1表达均减少,而Rb表达相应增加。
5.在MHCC97L皮下移植瘤模型中,H&E染色显示Lic5高低剂量组及联合顺铂治疗组裸鼠肝、肾、肺、脾等重要器官未发现明显组织结构异常。
6.在MHCC97L原位移植瘤模型中,与PBS空白对照组及小鼠IgG平行对照组比较,Lic5高低剂量治疗组生物发光信号均减弱,原位肿瘤体积明显小于对照组,差异具有统计学意义(p<0.05)。在PBS组及小鼠IgG对照组,肺转移率100%,在Lic5低剂量组(2.5mg/kg),肺转移率50%,在Lic5高剂量组(5mg/kg),肺转移率33.3%。结果与MHCC97L皮下移植瘤治疗结果一致。
结论：
1.抗人CDH17单克隆抗体Lic5能够显著抑制MHCC97L裸鼠皮下移植瘤及原位移植瘤的生长和肺转移,为肝癌的分子靶向治疗提供了有效的实验依据。
2.抗人CDH17单克隆抗体Lic5通过下调MHCC97L裸鼠皮下瘤组织中CDH17表达水平,失活Wnt/β-Catenin信号通路发挥抗肿瘤作用。
3.抗人CDH17单克隆抗体Lic5可以增加MHCC97L裸鼠皮下瘤细胞对顺铂的敏感性,为肿瘤的多药联合治疗提供了有效的实验依据。
4.抗人CDH17单克隆抗体Lic5在活体治疗中未发现明显生物学安全性隐患。
PartI
Preparation, purification and identification of monoclonal antibodies against human cadherin 17(CDH17)
Purpose:
Prepare Lic5 and Lic3 monoclonal antibodies (mAbs) from nude mouse ascites induced by Lic5 and Lic3 hybridoma cells and identify the purity and specificity of mAbs for the study of monoclonal antibody therapy in human hepatocellular carcinoma (HCC).
Materials and Methods:
Lic5 and Lic3 hybridoma cells were injected into abdominal cavity of BALB/c nu/nu mice and collected the ascites containing the mAbs. Purify Lic5 and Lic3 mAbs from mouse ascites by Protein A affinity chromatography and identify mAbs'purity and specificity by Western Blot and silver staining. Measure mAbs'concentration by Lowry method.
Results:
1. Lic5 and Lic3 hybridoma cells grew well in high activity. Ascites was collected 7-10 days after Lic5 hybridoma cell injection and 15 days after Lic3 hybridoma cell injection. The average yield of ascites with Lic5 and Lic3 hybridoma cells were 7.25ml and 5.5ml per nude mouse respectively.
2. From the silver staining of Lic5 and Lic3 mAbs, we only found two pieces of clear and independent bands at 50kD and 25kD, which represent the mAbs' heavy chain and light chain respectively. Western Blot detected CDH17 in cell lysate of MHCC97L and AGS with a single band at 120kD. Isotypes of Lic5 and Lic3 were identified by test paper and mAbs were IgG2b and IgG2a respectively.
3. Lic5 and Lic3 mAbs output of hybridoma ascites were 2.75mg/ml and 11.8mg/ml respectively.
Conclusion:
1. Mouse ascites containing Lic5 and Lic3 mAbs were harvested with high quality and high titer.
2. Lic5 and Lic3 mAbs against human CDH17 were prepared successfully with high purity and specificity.
3. Protein A affinity chromatography is suitable for purification of mAbs in the laboratory.
Part II
Characterization of changes in biological properties of human hepatocellualr carcinoma cell lines under the anti-CDH17 mAbs
Purpose:
To study the changes in cell proliferation, migration, invasion, colony formation and related drug sensitivity of human hepatoma cell lines MHCC97L, MHCC97H, PLC/PRF/5 and immortalized human hepatocyte MIHA under the Lic5 and Lic3 mAbs'treatment. To explore the possible underlying mechanisms.
Materials and Methods:
Detect the CDH17 protein and mRNA level in four cell lines such as MHCC97L, MHCC97H, PLC/PRF/5 and MIHA by the way of Western Blot and quantitative real-time PCR. Examine the binding capacity between Lic5 and Lic3 mAbs and CDH17 in the living cell membrane by the way of immunofluorescence staining. With the PBS and mouse IgG as the control, cell proliferation was detected by the way of MTT assay, cell migration was checked by wound healing assay, cell invasion assay was calculated by transwell method, cell colony formation was detected by plate colony assay. Drug sensitivity assay was monitored by MTT assay. Signaling pathways was explored by Western Blot.
Results:
1. CDH17 protein and mRNA were detected with a strongly high level in metastatic HCC cell lines MHCC97L, MHCC97H, but very little or undetectable level in primary HCC cell line PLC/PRF/5 and immortalized normal human hepatocyte cell line MIHA.
2. Lic5 and Lic3 mAbs can bind with the CDH17 in the membrane of living cell lines MHCC9L and MHCC97H.
3. Cell proliferation ability of MHCC97L and MHCC97H in mAbs'groups was significantly weakened. The statistic analysis showed significant difference between mAbs'groups and controls (p<0.05). There was no difference between mAbs'groups and controls in PLC/PRF/5, and MIHA cell lines.
4. The number of clone formation, migration and invasion in Lic5 and Lic3 mAbs' groups of MHCC97L and MHCC97H cells was significantly less than that in PBS and mouse Ig groups. Statistic analysis showed significant difference between mAbs'and control groups.
5. The drug sensitivity and inhibition rate of cisplatin to MHCC97L and MHCC97H cells was significantly elevated as compared to PBS and mouse Ig controls.
6. Lic5 and Lic3 mAbs can downregulate the CDH17 protein expression level in MHCC97L and MHCC97H cells and had no any effect on CDH17 mRNA level.
7. Lic5 and Lic3 mAbs can downregulate theβ-catenin protein expression level in MHCC97L and MHCC97H cells, which was the key molecule in Wnt/p-catenin signaling pathway.
Conclusion:
1. Lic5 and Lic3 mAbs can bind with CDH17 in cell membrane in living MHCC97L and MHCC97H cells.
2. The MHCC97L and MHCC97H cells in mAs'groups showed great changes in the cell biological properties as compared to PBS and mouse Ig controls including retarded proliferation; decreased ability in cell clone formation, cell migration and invasion.
3. Lic5 and Lic3 mAbs increased the drug sensititvity of MHCC97L and MHCC97H to cisplatin significantly.
4. The CDH17 andβ-catenin protein level were significantly reduced by Lic5 and Lic3 mAbs in MHCC97L and MHCC97H cells.
Part III
The inhibition role of anti-CDH17 monoclonal antibody and combination with cisplatin on hepatocellular carcinoma in vivo and the exploration of the anti-tumor mechanisms.
Purpose:
To studay the anti-tumor effect of Lic5 and the combination between Lic5 and cisplatin in subcutaneous xenograft model and orthotopic transplantation model of MHCC97L cell. To explore the underlying signaling pathway related to the mAbs' therapy.
Materials and Methods:
Use MHCC97L or with luciferase transfection to establish the subcutaneous tumor model and orthotopic tansplantation tumor model of human HCC in nude mouse. The subcutaneous tumor models in nude mice were divided into six groups, PBS empty control group, Mouse IgG parallel control group, Lic5 mAb low dosage group(2.5mg/kg), Lic5 mAb high dosage group(5mg/kg), cisplatin group(lmg/kg), the combination group of Lic5 mAb and cisplatin(Lic55mg/kg, cisplatin lmg/kg). Observe the impact of Lic5 and the combination with cisplatin on subcutaneous tumor growth. Detect the change of expression level of CDH17 and key molecules in related signaling pathway by the way of Western Blot. The orthotopic transplantation tumor models in nude mice were divided into four groups, PBS empty control group, Mouse IgG parallel control group, Lic5 mAb low dosage group(2.5mg/kg), Lic5 mAb high dosage group(5mg/kg). Use the bioluminescence to montitor the tumr growth in different groups.
Results:
1. Compared with PBS and mouse Ig control in subcutaneous model, the tumor growth was remarkably inhibited in mAbs'groups. Among the former three groups, the tumor growth in Lic5 and cisplatin combination group showed the strongest inhibition. Statistics analysis showed significant difference between the mAbs'and control gourps.
2. In the subcutaneous tumor models, the lung metastasis rate in PBS and mouse Ig groups were all 83.3%, Lic5 low dosage, high dosage and combination groups were 50%,33%and 0.
3. The number of Ki67 positive cells were significantly decreased in mAbs'groups compared with PBS and mouse Ig groups in subcutaneous tumor models. Statistics analysis showed significant difference between mAbs'and control groups.
4. Western Blot showed the expression of CDH17 was significantly downregulated in mAbs'treatment groups whereas high expression of CDH17 in control groups. The key molecules in Wnt/β-catenin, (3-catenin and Cyclin D1 were also downregulated in mAbs'treatment groups, but Rb was upregulated in the same groups.
5. There was no obvious tissue abnormalities in liver, kidney, lung and spleen organs in mAbs'treatment groups.
6. Compared with the PBS and moue Ig controls in orthotopic tumor models, bioluminescence signals and tumor volume were reduced significantly in mAs' treatment groups. Statistics analysis showed significant difference between mAbs' groups and control groups. The tumor lung metastasis rate in PBS and mouse Ig groups were all 100%and 50%,33.3%in Lic5 low and high dosage groups.
Conclusion:
1. Anti-CDH17 monoclonal antibody Lic5 can significantly inhibit the MHCC97L tumor growth and lung metastasis in nude mouse model and is a promising candidate in HCC targeted therapy.
2. Lic5 mAb inhibited MHCC97L tumor growth by downregulation of CDH17 and inactivation of Wnt/β-catenin signaling pathway.
3. Lic5 mAb can improve the drug sensitivity to cisplatin in MHCC97L in vivo and provide an effective experimental basis for the multi-drug combination therapy in HCC.
4. No risk in biological safety had been observed during the period of mAb administration.
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