逆转录病毒载体表达的TGFα反义RNA对人脑胶质瘤细胞BT325的生长抑制和诱导分化作用
摘要
转化生化因子α(TGFα)是一种小肽分子，正常细胞较少产生，而在许多肿瘤组织(肾癌，黑色素瘤等)却大量合成。TGFα与细胞表面的表皮生长因子受体(EGFR)结合，以自分泌方式参与肿瘤的形成和生长，具有重要的肿瘤诊断、预后及疗效评估意义。胶质瘤是临床常见的肿瘤之一，其死亡率极高。有关胶质瘤中TGFα状况的文献报道较少且互有出入。采用分子生物学方法研究胶质瘤中TGFα及相关基因的改变并予以纠正具有重要的理论和临床意义。
为开展此项工作，我们函索到phTGF-1-10-925质粒，其载体部分为pBR327，在EcoRI位点处插有925bp的TGFα cDNA片段。为鉴定此质粒，先用EcoRI及EcoRIPstl进行酶切图谱分析，此外，我们合成了一对针对TGFα第四外显子的引物，以该质粒为模板进行PCR，并对PCR片段进行DNA序列分析。结果表明所扩增片段确系TGFα第四外显子。
我们以phTGFα-1-10-925和c-myc质粒探针，检测了两株瘤细胞系(BT325，SHG44)及数例临床脑胶质瘤标本中TGFα和c-myc基因的变化。Southern杂交表明TGFα基因无扩增，但两株细胞系及4／6病例有TGFα基因的高表达，正常脑组织无表达。本文还以建立的定量差异PCR技术(靶基因为TGFα第四外显子，参比基因为ras第一外显子)考察了胶瘤中TGFα基因的拷贝数，其结果与Southern杂交相同。c-myc基因检测表明各种胶质瘤中有不同程度的c-myc基因扩增和重排，同时伴有c-myc基因的高表达。TGFα和c-myc的高表达具有平行性。
我们采用化学合成方法合成了TGFαN末端七肽小分子(Val-Val-Ser-His-Phe-Asn-Asp)，并以EDC为交联剂将其与BSA偶联后免疫兔子制备出TGFα抗体。该抗体抗同一抗原决定簇，与EGF无交叉反应，且具有抑制BT325细胞生长的活性。
Transforming growth factorα (TGFα) is a polypeptide growth factor produced mainly by a variety of malignant cel types ( renal carcinoma, melanoma, etc.) and known to bind the epidermal growth factor receptor (EGFR) on cell surface in due form of autocrine mechanism. Elevation of the TGFα in body fluids may suggest the possible presence of tumor growth. However, the pathological significance of TGFα in human glioma seems to be paradoxical in so far reported literatures. Therefore. it is important to find out further any changes in TGFα and related genes in glioma and to correct them by biotechnological approaches for therapeutic purpose. I
In this report, a plasmid phTGF-1 —10—925. consisting of pBR327 vector
with a 925 bp TGFα cDNA fragment cloned at its EcoRI site, was first digested with EcoRI and EcoRI plus PstI respectively. This plasmid, proved to be correct by the restriction pattern was taken as a template for the polymerase chain reaction (PCR) of TGFα -IV exon fragment. DNA sequencing of the PCR product demonstrated the fidelity of the amplified fragment.
Using phTGF-1-10-925 and phc-myc as probes, the gene changes of TGFα and c-myc in two glioma cell lines, BT325 and SHG44, as well as several surgical specimens of human glioma were observed. The results indicated that TGFα gene was overexpressed rather than amplified in glioma, whereas there were amplification, rearrangement, and overexpression of c-myc protooncogene. The expression levels of these two genes were shown to be parallel.
A N-terminal hepta-peptide of TGFα(VVSHFND) was chemically synthesized and coupled with BSA to form a complete antigen. Antibodies were prepared by immunizing rabbits with this conjugate, and immunoassay showed that the immunoabsorbed antiserum recognized only the single epitope of hepta-peptide. No cross reaction with EGF was observed. By adding this antiserum into cell
为开展此项工作，我们函索到phTGF-1-10-925质粒，其载体部分为pBR327，在EcoRI位点处插有925bp的TGFα cDNA片段。为鉴定此质粒，先用EcoRI及EcoRIPstl进行酶切图谱分析，此外，我们合成了一对针对TGFα第四外显子的引物，以该质粒为模板进行PCR，并对PCR片段进行DNA序列分析。结果表明所扩增片段确系TGFα第四外显子。
我们以phTGFα-1-10-925和c-myc质粒探针，检测了两株瘤细胞系(BT325，SHG44)及数例临床脑胶质瘤标本中TGFα和c-myc基因的变化。Southern杂交表明TGFα基因无扩增，但两株细胞系及4／6病例有TGFα基因的高表达，正常脑组织无表达。本文还以建立的定量差异PCR技术(靶基因为TGFα第四外显子，参比基因为ras第一外显子)考察了胶瘤中TGFα基因的拷贝数，其结果与Southern杂交相同。c-myc基因检测表明各种胶质瘤中有不同程度的c-myc基因扩增和重排，同时伴有c-myc基因的高表达。TGFα和c-myc的高表达具有平行性。
我们采用化学合成方法合成了TGFαN末端七肽小分子(Val-Val-Ser-His-Phe-Asn-Asp)，并以EDC为交联剂将其与BSA偶联后免疫兔子制备出TGFα抗体。该抗体抗同一抗原决定簇，与EGF无交叉反应，且具有抑制BT325细胞生长的活性。
Transforming growth factorα (TGFα) is a polypeptide growth factor produced mainly by a variety of malignant cel types ( renal carcinoma, melanoma, etc.) and known to bind the epidermal growth factor receptor (EGFR) on cell surface in due form of autocrine mechanism. Elevation of the TGFα in body fluids may suggest the possible presence of tumor growth. However, the pathological significance of TGFα in human glioma seems to be paradoxical in so far reported literatures. Therefore. it is important to find out further any changes in TGFα and related genes in glioma and to correct them by biotechnological approaches for therapeutic purpose. I
In this report, a plasmid phTGF-1 —10—925. consisting of pBR327 vector
with a 925 bp TGFα cDNA fragment cloned at its EcoRI site, was first digested with EcoRI and EcoRI plus PstI respectively. This plasmid, proved to be correct by the restriction pattern was taken as a template for the polymerase chain reaction (PCR) of TGFα -IV exon fragment. DNA sequencing of the PCR product demonstrated the fidelity of the amplified fragment.
Using phTGF-1-10-925 and phc-myc as probes, the gene changes of TGFα and c-myc in two glioma cell lines, BT325 and SHG44, as well as several surgical specimens of human glioma were observed. The results indicated that TGFα gene was overexpressed rather than amplified in glioma, whereas there were amplification, rearrangement, and overexpression of c-myc protooncogene. The expression levels of these two genes were shown to be parallel.
A N-terminal hepta-peptide of TGFα(VVSHFND) was chemically synthesized and coupled with BSA to form a complete antigen. Antibodies were prepared by immunizing rabbits with this conjugate, and immunoassay showed that the immunoabsorbed antiserum recognized only the single epitope of hepta-peptide. No cross reaction with EGF was observed. By adding this antiserum into cell
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