血小板源性生长因子-B（PDGF-B）信号通路在胃癌转移中的作用机制的实验研究
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摘要
第一部分PDGF-B和PDGFR-β在胃癌组织的表达及其临床意义
目的：探讨血小板源性生长因子B（PDGF-B）和血小板源性生长因子受体β(PDGFR-β)在胃癌组织中的表达情况及其临床意义。
方法：采用免疫组化SP法检测32例胃癌手术标本中胃癌组织及其对应的正常胃粘膜组织中PDGF-B和PDGFR-β的表达情况，并与胃癌患者临床资料进行相关性分析。
结果：胃癌组织中PDGF-B主要表达于肿瘤细胞，阳性率70.3%(45/64)，PDGFR-β主要表达于肿瘤间质细胞，阳性率60.9%(39/64)；均明显高于正常的胃粘膜组织中的PDGF-B阳性率4.7%(3/64)和PDGFR-β的阳性率3.1%(2/64)(P<0.05)。PDGF-B和PDGFR-β的表达与肿瘤浸润深度，淋巴结转移及肿瘤分期呈正相关(P<0.05)；与肿瘤分化程度没有相关性(P>0.05)。
结论：PDGF-B和PDGFR-β在胃癌组织中高表达并与胃癌的进展及转移关系密切；检测PDGF-B和PDGFR-β的表达可以作为判断胃癌转移和预后的指标之一。
第二部分：PDGF-B慢病毒载体的构建、包装和PDGF-B过表达胃癌细胞株的构建
目的：构建PDGF-B的慢病毒载体pLeno-DCE-PDGF-B并转染成功构建PDGF-B稳定过表达的胃癌细胞。
方法：构建PDGF-B慢病毒载体pLeno-DCE-PDGF-B，感染293T细胞，收集上清液测定病毒滴度。用慢病毒转染SGC7901和BGC823胃癌细胞株，用免疫荧光观察和western-blot方法鉴定PDGF-B稳定过表达胃癌细胞构建结果。
结果：通过PCR及酶切鉴定成功构建pLeno-DCE-PDGF-B载体，包装并得到高滴度的病毒颗粒。用慢病毒转染后SGC7901和BGC823胃癌细胞免疫荧光观察呈绿色荧光，转染后SGC7901和BGC823胃癌细胞中PDGF-B蛋白表达明显高于常规SGC7901和BGC823胃癌细胞（P<0.05）。
结论：用慢病毒构建方法可成功构建并包装得到高滴度的PDGF-B慢病毒颗粒；采用PDGF-B慢病毒转染可成功构建PDGF-B稳定过表达的胃癌细胞。
第三部分PDGF-B过表达对胃癌细胞生长，侵袭能力的影响及其机制
目的：探讨PDGF-B过表达对胃癌细胞株生长和侵袭能力的影响及其机制。
方法：慢病毒载体转染构建PDGF-B稳定过表达胃癌细胞株。用MTT法和Transwell侵袭实验分别检测PDGF-B稳定过表达SGC7901和BGC823胃癌细胞及常规SGC7901和BGC823胃癌细胞的生长和侵袭能力，并进行相互比较分析。采用Western-blot法检测PDGF-B过表达前后AKT-1和E-cadherin蛋白在SGC7901和BGC823胃癌细胞中的表达情况。
结果：PDGF-B过表达SGC7901和BGC823胃癌细胞的生长曲线明显高于常规SGC7901和BGC823胃癌细胞(P<0.05)。PDGF-B过表达SGC7901和BGC823胃癌细胞的侵袭数量分别为65.4±2.4和70.2±3.2，明显多于常规SGC7901和BGC823胃癌细胞的21.6±1.6和30.2±2.2(P<0.05)。PDGF-B过表达SGC7901和BGC823胃癌细胞中AKT-1蛋白表达明显高于常规SGC7901和BGC823胃癌细胞(P<0.05)，E-cadherin蛋白在PDGF-B过表达SGC7901和BGC823胃癌细胞和常规SGC7901和BGC823胃癌细胞中的表达没有明显差异(P>0.05)。
结论：PDGF-B过表达可以明显增强胃癌细胞的生长和侵袭能力，可能与胃癌进展和转移相关。其机制可能与PDGF-B自分泌作用激活PI3K/AKT通路相关。
第四部分PDGF-B信号通路与上皮间质化和胃癌转移的相关性及其机制
目的：探讨PDGF-B信号通路与上皮间质化和胃癌转移的相关性及其作用机制。
方法：采用Western-blot方法检测PDGF-B过表达前后SGC7901和BGC823胃癌细胞中AKT-1、ERK-1、E-cadherin和N-cadherin的蛋白表达情况，并进行比较分析。采用细胞共培养技术模拟激活PDGF-B信号通路，用Western-blot和RT-PCR方法检测共培养情况下常规SGC7901和BGC823胃癌细胞，共培养情况下PDGF-B过表达SGC7901和BGC823胃癌细胞中AKT-1、ERK-1、E-cadherin和N-cadherin蛋白及mRNA表达情况，并进行比较分析。
结果：PDGF-B过表达前后SGC7901和BGC823胃癌细胞中ERK-1、E-cadherin和N-cadherin蛋白表达没有明显差异(P>0.05)；PDGF-B过表达SGC7901和BGC823胃癌细胞中AKT-1蛋白表达明显高于常规SGC7901和BGC823胃癌细胞(P<0.05)。共培养的PDGF-B过表达SGC7901和BGC823胃癌细胞中AKT-1和ERK-1蛋白及mRNA的表达明显高于常规SGC7901和BGC823胃癌细胞(P<0.05)。上皮间质化相关指标E-cadherin蛋白及mRNA在共培养的PDGF-B过表达SGC7901和BGC823胃癌细胞中的表达明显低于常规SGC7901和BGC823胃癌细胞(P<0.05)；上皮间质化相关指标N-cadherin蛋白及mRNA在PDGF-B过表达SGC7901和BGC823胃癌细胞中的表达明显高于常规SGC7901和BGC823胃癌细胞(P<0.05)。常规SGC7901和BGC823胃癌细胞共培养前后AKT-1、ERK-1、N-cadherin和E-cadherin的蛋白表达没有明显差异(P>0.05)。PDGF-B过表达SGC7901和BGC823胃癌细胞共培养前后AKT-1蛋白表达没有明显差异(P>0.05)，PDGF-B过表达SGC7901和BGC823胃癌细胞共培养前后ERK-1、E-cadherin和N-cadherin蛋白表达有明显差异(P<0.05)。
结论：PDGF-B信号通过自分泌作用不能诱导胃癌细胞发生上皮间质化。PDGF-B信号可以通过旁分泌作用并与肿瘤间质细胞相互作用促进胃癌细胞发生上皮间质化，从而促进胃癌进展和转移；其机制可能与PDGF-B信号通路下游的MAPK/ERK通路的激活和肿瘤微环境联合作用诱发胃癌细胞上皮间质化有关。PDGF-B信号通路可能是胃癌有效的治疗靶点之一。
PART I CLINICOPATHOLOGICAL SIGNIFICANCE OFPDGF-B AND PLATELET-DERIVED GROWTH FACTORRECPERTOR-β EXPRESSION IN GASTRIC CARCINOMA
Objective: To observe the relationships between expressions ofplatelet-derived growth factor B(PDGF-B), platelet-derived growth factorreceptor β(PDGFR-β) and clinicolpathological features of patients withgastric carcinoma.
Methods: A series of32gastric carcinoma cases that had undergonesurgical resection was collected. Gastric carcinoma tissues and normalgastric mucosa tissues were examined immunohistochemically usingantibodies against PDGF-B and PDGFR-β, followed by further analysisabout the correlation between PDGF-B and PDGFR-β expression and theclinicolpathological features of patients.
Results: In surgical specimens, tumor cells expressed PDGF-B,positive rate was70.3%(45/64); but PDGFR-β was expressedpredominantly by tumor stromal cells, positive rate was60.9%(39/64); both
of them were much higher than those of in normal gastric mucosa
tissues(4.7%,3/64and3.1%,2/64respectively). Expressions of PDGF-B
and PDGFR-β were positively correlated with the depth of cancer invasion,
lymph node metastasis and tumor-node-metastasis(TNM) stage(P<0.05);but expressions of PDGF-B and PDGFR-β were no correlated with thetumor cell differentiation(P>0.05).
Conclusions: Our data indicate that expressions of PDGF-B andPDGFR-β were much higher in gastric carcinoma tissue and were correlatedwith cancer progression and lymphogenous metastasis of gastric carcinoma;suggest that detection of PDGF-B and PDGFR-β were important markers forjudgment of tumor metastasis and prognosis of gastric carcinoma.
PART II CONSTRUCTION OF LENTIVIRAL VECTORPLENO-DCE-PDGF-B AND STABLE PDGF-BOVEREXPRESSION GASTRIC CARCINOMA CELL LINE
Objective: To construct a lentiviral vector that stably expressplatelet-derived growth factor B (PDGF-B) and to construct stable PDGF-Boverexpression gastric carcinoma cell line by lentiviral vector transfection.
Methods: The lentiviral vector pLeno-DCE-PDGF-B was constructedand transfected into293T cells. The supernatant containing the lentivirusparticles was harvested to determine the virus titer and high titer lentivirusparticles was gathered. Then PDGF-B lentiviral vector was transfected intoSGC7901and BGC823gastric carcinoma cells for construction of stablePDGF-B overexpression gastric carcinoma cell lines. Western-blot andimmunofluorescence were used for evaluation of the construction of stablePDGF-B overexpression gastric carcinoma cell lines.
Results: The lentiviral vector was correctly constructed and verified bysequencing. High titer PDGF-B lentiviral vector was acquired successfully.After transfection, two gastric carcinoma cell line SGC7901and BGC823were green color by detection of fluorescence microscope; and theexpression of PDGF-B protein in transfected SGC7901and BGC823gastric carcinoma cells were much higher than that of in normal SGC7901and BGC823gastric carcinoma cells(P<0.05).
Conclusion: We have successfully constructed PDGF-B lentiviralvector and acquired high titer PDGF-B lentiviral vector. Lentiviral vectortransfection was an effective method for construction of stable PDGF-Boverexpression gastric carcinoma cell lines.
PART III THE INFLUENCE ON GROWTH ANDINVASION OF GASTRIC CARCINOMA CELL CAUSEDBY PDGF-B OVEREXPRESSION AND ITS MECHANISM
Objective: To observe the influence on growth and invasion of gastriccarcinoma cell caused by PDGF-B overexpression and to approach themechanism about how PDGF-B to affect the growth and invasion of gastriccarcinoma cells.
Methods: Stable PDGF-B overexpression gastric carcinoma cell lineswere constructed. MTT assay was used for detection of cell growth andtranswell invasion assay was used for detection of invasion for both PDGF-Boverexpression and normal SGC7901and BGC823gastric carcinoma cells.Western-blot was used for detection of expression of AKT-1and E-cadherinbefore and after transfection to approach the mechanism.
Results: Growth curve of PDGF-B overexpression SGC7901andBGC823gastric carcinoma cells were much higher than those of normalSGC7901and BGC823gastric carcinoma cells(P<0.05). Invasion cellnumber of PDGF-B overexpression SGC7901and BGC823gastriccarcinoma cells(65.4±2.4and70.2±3.2, respectively) were much higher thanthose of normal SGC7901and BGC823gastric carcinoma cells(21.6±1.6and30.2±2.2, respectively)(P<0.05). Expression of AKT-1in PDGF-B
overexpression SGC7901and BGC823gastric carcinoma cells were much
higher than normal SGC7901and BGC823gastric carcinoma cells(P<0.05).
Expression of E-cadherin were no difference between PDGF-B
overexpression and normal SGC7901and BGC823gastric carcinoma
cells(P>0.05).
Conclusions: Overexpression of PDGF-B increases the growth andinvasion of gastric carcinoma cells so as to promote tumor metastasis.Activation of PI3K/AKT signal pathway by PDGF-B autocrine might leadthe increases of the growth and invasion of gastric carcinoma cells.
PART Ⅳ THE RELATIONSHIP BETWEEN PDGF-BSIGNALING, EPITHELIAL–MESENCHYMALTRANSIYION AND METASTASIS OF GASTRICCARCINOMA AND ITS MECHANISM
Objective: To approach the relationships between PDGF-B signaling，epithelial–mesenchymal transition(EMT) and metastasis of gastriccarcinoma and to investigate the mechanism of PDGF-B signaling inmetastasis of gastric carcinoma.
Methods: Expressions of AKT-1, ERK-1, E-cadherin and N-cadherinprotein in SGC7901and BGC823gastric carcinoma cells before and afterPDGF-B transfection were detected by western-blot. Cell coculture wasused for activation of PDGF-B signaling. RT-PCR was used for detection ofmRNA of AKT-1, ERK-1, E-cadherin and N-cadherin in coculture PDGF-Boverexpression SGC7901and BGC823gastric carcinoma cells and incoculture normal SGC7901and BGC823gastric carcinoma cells, alsowestern-blot was used for detection of protein of AKT-1, ERK-1, E-cadherinand N-cadherin in coculture PDGF-B overexpression SGC7901andBGC823gastric carcinoma cells and in coculture normal SGC7901andBGC823gastric carcinoma cells.
Results: There were no differences between expressions of E-cadherin,N-cadherin and ERK-1protein in SGC7901and BGC823gastric carcinomacells before and after PDGF-B overexpression(P>0.05); the expressions ofAKT-1protein in PDGF-B overexpression SGC7901and BGC823gastriccarcinoma cells were much higher than those of in normal SGC7901andBGC823gastric carcinoma cells(P<0.05). The expressions of mRNA andprotein of AKT-1and ERK-1in cocultured PDGF-B overexpressionSGC7901and BGC823gastric carcinoma cells were much higher than thoseof in cocultured normal SGC7901and BGC823gastric carcinomacells(P<0.05). Expressions of mRNA and protein of EMT-related E-cadherinin coculture PDGF-B overexpression SGC7901and BGC823gastric carcinoma cells were much lower than those of in cocultured normalSGC7901and BGC823gastric carcinoma cells(P<0.05), and expressions ofmRNA and protein of ETM-related N-cadherin in cocultured PDGF-Boverexpression SGC7901and BGC823gastric carcinoma cells were muchhigher than those of in cocultured normal SGC7901and BGC823gastriccarcinoma cells(P<0.05). There were no differences between expressions ofAKT-1, ERK-1, E-cadherin and N-cadherin protein in normal SGC7901and BGC823gastric carcinoma cells before and after coculture(P>0.05).There were no differences between expressions of AKT-1protein inPDGF-B overexpression SGC7901and BGC823gastric carcinoma cellsbefore and after coculture(P>0.05); there were apparently differencesbetween expressions E-cadherin, N-cadherin and ERK-1protein inPDGF-B overexpression SGC7901and BGC823gastric carcinoma cellsbefore and after coculture(P<0.05).
Conclusions: PDGF-B signaling might not induce EMT in gastriccarcinoma cells through autocrine. PDGF-B signaling might induce EMT ingastric carcinoma cells through paracrine and joint action with tumorstromal cells so as to promote metastasis of gastric carcinoma. BothMAPK/ERK signal pathway(a downstream targets of PDGF signalingpathway) and tumor microenvironment might participate in the process ofEMT. Consequently, PDGF-B signaling might be an effective therapy targetfor gastric carcinoma.
目的：探讨血小板源性生长因子B（PDGF-B）和血小板源性生长因子受体β(PDGFR-β)在胃癌组织中的表达情况及其临床意义。
方法：采用免疫组化SP法检测32例胃癌手术标本中胃癌组织及其对应的正常胃粘膜组织中PDGF-B和PDGFR-β的表达情况，并与胃癌患者临床资料进行相关性分析。
结果：胃癌组织中PDGF-B主要表达于肿瘤细胞，阳性率70.3%(45/64)，PDGFR-β主要表达于肿瘤间质细胞，阳性率60.9%(39/64)；均明显高于正常的胃粘膜组织中的PDGF-B阳性率4.7%(3/64)和PDGFR-β的阳性率3.1%(2/64)(P<0.05)。PDGF-B和PDGFR-β的表达与肿瘤浸润深度，淋巴结转移及肿瘤分期呈正相关(P<0.05)；与肿瘤分化程度没有相关性(P>0.05)。
结论：PDGF-B和PDGFR-β在胃癌组织中高表达并与胃癌的进展及转移关系密切；检测PDGF-B和PDGFR-β的表达可以作为判断胃癌转移和预后的指标之一。
第二部分：PDGF-B慢病毒载体的构建、包装和PDGF-B过表达胃癌细胞株的构建
目的：构建PDGF-B的慢病毒载体pLeno-DCE-PDGF-B并转染成功构建PDGF-B稳定过表达的胃癌细胞。
方法：构建PDGF-B慢病毒载体pLeno-DCE-PDGF-B，感染293T细胞，收集上清液测定病毒滴度。用慢病毒转染SGC7901和BGC823胃癌细胞株，用免疫荧光观察和western-blot方法鉴定PDGF-B稳定过表达胃癌细胞构建结果。
结果：通过PCR及酶切鉴定成功构建pLeno-DCE-PDGF-B载体，包装并得到高滴度的病毒颗粒。用慢病毒转染后SGC7901和BGC823胃癌细胞免疫荧光观察呈绿色荧光，转染后SGC7901和BGC823胃癌细胞中PDGF-B蛋白表达明显高于常规SGC7901和BGC823胃癌细胞（P<0.05）。
结论：用慢病毒构建方法可成功构建并包装得到高滴度的PDGF-B慢病毒颗粒；采用PDGF-B慢病毒转染可成功构建PDGF-B稳定过表达的胃癌细胞。
第三部分PDGF-B过表达对胃癌细胞生长，侵袭能力的影响及其机制
目的：探讨PDGF-B过表达对胃癌细胞株生长和侵袭能力的影响及其机制。
方法：慢病毒载体转染构建PDGF-B稳定过表达胃癌细胞株。用MTT法和Transwell侵袭实验分别检测PDGF-B稳定过表达SGC7901和BGC823胃癌细胞及常规SGC7901和BGC823胃癌细胞的生长和侵袭能力，并进行相互比较分析。采用Western-blot法检测PDGF-B过表达前后AKT-1和E-cadherin蛋白在SGC7901和BGC823胃癌细胞中的表达情况。
结果：PDGF-B过表达SGC7901和BGC823胃癌细胞的生长曲线明显高于常规SGC7901和BGC823胃癌细胞(P<0.05)。PDGF-B过表达SGC7901和BGC823胃癌细胞的侵袭数量分别为65.4±2.4和70.2±3.2，明显多于常规SGC7901和BGC823胃癌细胞的21.6±1.6和30.2±2.2(P<0.05)。PDGF-B过表达SGC7901和BGC823胃癌细胞中AKT-1蛋白表达明显高于常规SGC7901和BGC823胃癌细胞(P<0.05)，E-cadherin蛋白在PDGF-B过表达SGC7901和BGC823胃癌细胞和常规SGC7901和BGC823胃癌细胞中的表达没有明显差异(P>0.05)。
结论：PDGF-B过表达可以明显增强胃癌细胞的生长和侵袭能力，可能与胃癌进展和转移相关。其机制可能与PDGF-B自分泌作用激活PI3K/AKT通路相关。
第四部分PDGF-B信号通路与上皮间质化和胃癌转移的相关性及其机制
目的：探讨PDGF-B信号通路与上皮间质化和胃癌转移的相关性及其作用机制。
方法：采用Western-blot方法检测PDGF-B过表达前后SGC7901和BGC823胃癌细胞中AKT-1、ERK-1、E-cadherin和N-cadherin的蛋白表达情况，并进行比较分析。采用细胞共培养技术模拟激活PDGF-B信号通路，用Western-blot和RT-PCR方法检测共培养情况下常规SGC7901和BGC823胃癌细胞，共培养情况下PDGF-B过表达SGC7901和BGC823胃癌细胞中AKT-1、ERK-1、E-cadherin和N-cadherin蛋白及mRNA表达情况，并进行比较分析。
结果：PDGF-B过表达前后SGC7901和BGC823胃癌细胞中ERK-1、E-cadherin和N-cadherin蛋白表达没有明显差异(P>0.05)；PDGF-B过表达SGC7901和BGC823胃癌细胞中AKT-1蛋白表达明显高于常规SGC7901和BGC823胃癌细胞(P<0.05)。共培养的PDGF-B过表达SGC7901和BGC823胃癌细胞中AKT-1和ERK-1蛋白及mRNA的表达明显高于常规SGC7901和BGC823胃癌细胞(P<0.05)。上皮间质化相关指标E-cadherin蛋白及mRNA在共培养的PDGF-B过表达SGC7901和BGC823胃癌细胞中的表达明显低于常规SGC7901和BGC823胃癌细胞(P<0.05)；上皮间质化相关指标N-cadherin蛋白及mRNA在PDGF-B过表达SGC7901和BGC823胃癌细胞中的表达明显高于常规SGC7901和BGC823胃癌细胞(P<0.05)。常规SGC7901和BGC823胃癌细胞共培养前后AKT-1、ERK-1、N-cadherin和E-cadherin的蛋白表达没有明显差异(P>0.05)。PDGF-B过表达SGC7901和BGC823胃癌细胞共培养前后AKT-1蛋白表达没有明显差异(P>0.05)，PDGF-B过表达SGC7901和BGC823胃癌细胞共培养前后ERK-1、E-cadherin和N-cadherin蛋白表达有明显差异(P<0.05)。
结论：PDGF-B信号通过自分泌作用不能诱导胃癌细胞发生上皮间质化。PDGF-B信号可以通过旁分泌作用并与肿瘤间质细胞相互作用促进胃癌细胞发生上皮间质化，从而促进胃癌进展和转移；其机制可能与PDGF-B信号通路下游的MAPK/ERK通路的激活和肿瘤微环境联合作用诱发胃癌细胞上皮间质化有关。PDGF-B信号通路可能是胃癌有效的治疗靶点之一。
PART I CLINICOPATHOLOGICAL SIGNIFICANCE OFPDGF-B AND PLATELET-DERIVED GROWTH FACTORRECPERTOR-β EXPRESSION IN GASTRIC CARCINOMA
Objective: To observe the relationships between expressions ofplatelet-derived growth factor B(PDGF-B), platelet-derived growth factorreceptor β(PDGFR-β) and clinicolpathological features of patients withgastric carcinoma.
Methods: A series of32gastric carcinoma cases that had undergonesurgical resection was collected. Gastric carcinoma tissues and normalgastric mucosa tissues were examined immunohistochemically usingantibodies against PDGF-B and PDGFR-β, followed by further analysisabout the correlation between PDGF-B and PDGFR-β expression and theclinicolpathological features of patients.
Results: In surgical specimens, tumor cells expressed PDGF-B,positive rate was70.3%(45/64); but PDGFR-β was expressedpredominantly by tumor stromal cells, positive rate was60.9%(39/64); both
of them were much higher than those of in normal gastric mucosa
tissues(4.7%,3/64and3.1%,2/64respectively). Expressions of PDGF-B
and PDGFR-β were positively correlated with the depth of cancer invasion,
lymph node metastasis and tumor-node-metastasis(TNM) stage(P<0.05);but expressions of PDGF-B and PDGFR-β were no correlated with thetumor cell differentiation(P>0.05).
Conclusions: Our data indicate that expressions of PDGF-B andPDGFR-β were much higher in gastric carcinoma tissue and were correlatedwith cancer progression and lymphogenous metastasis of gastric carcinoma;suggest that detection of PDGF-B and PDGFR-β were important markers forjudgment of tumor metastasis and prognosis of gastric carcinoma.
PART II CONSTRUCTION OF LENTIVIRAL VECTORPLENO-DCE-PDGF-B AND STABLE PDGF-BOVEREXPRESSION GASTRIC CARCINOMA CELL LINE
Objective: To construct a lentiviral vector that stably expressplatelet-derived growth factor B (PDGF-B) and to construct stable PDGF-Boverexpression gastric carcinoma cell line by lentiviral vector transfection.
Methods: The lentiviral vector pLeno-DCE-PDGF-B was constructedand transfected into293T cells. The supernatant containing the lentivirusparticles was harvested to determine the virus titer and high titer lentivirusparticles was gathered. Then PDGF-B lentiviral vector was transfected intoSGC7901and BGC823gastric carcinoma cells for construction of stablePDGF-B overexpression gastric carcinoma cell lines. Western-blot andimmunofluorescence were used for evaluation of the construction of stablePDGF-B overexpression gastric carcinoma cell lines.
Results: The lentiviral vector was correctly constructed and verified bysequencing. High titer PDGF-B lentiviral vector was acquired successfully.After transfection, two gastric carcinoma cell line SGC7901and BGC823were green color by detection of fluorescence microscope; and theexpression of PDGF-B protein in transfected SGC7901and BGC823gastric carcinoma cells were much higher than that of in normal SGC7901and BGC823gastric carcinoma cells(P<0.05).
Conclusion: We have successfully constructed PDGF-B lentiviralvector and acquired high titer PDGF-B lentiviral vector. Lentiviral vectortransfection was an effective method for construction of stable PDGF-Boverexpression gastric carcinoma cell lines.
PART III THE INFLUENCE ON GROWTH ANDINVASION OF GASTRIC CARCINOMA CELL CAUSEDBY PDGF-B OVEREXPRESSION AND ITS MECHANISM
Objective: To observe the influence on growth and invasion of gastriccarcinoma cell caused by PDGF-B overexpression and to approach themechanism about how PDGF-B to affect the growth and invasion of gastriccarcinoma cells.
Methods: Stable PDGF-B overexpression gastric carcinoma cell lineswere constructed. MTT assay was used for detection of cell growth andtranswell invasion assay was used for detection of invasion for both PDGF-Boverexpression and normal SGC7901and BGC823gastric carcinoma cells.Western-blot was used for detection of expression of AKT-1and E-cadherinbefore and after transfection to approach the mechanism.
Results: Growth curve of PDGF-B overexpression SGC7901andBGC823gastric carcinoma cells were much higher than those of normalSGC7901and BGC823gastric carcinoma cells(P<0.05). Invasion cellnumber of PDGF-B overexpression SGC7901and BGC823gastriccarcinoma cells(65.4±2.4and70.2±3.2, respectively) were much higher thanthose of normal SGC7901and BGC823gastric carcinoma cells(21.6±1.6and30.2±2.2, respectively)(P<0.05). Expression of AKT-1in PDGF-B
overexpression SGC7901and BGC823gastric carcinoma cells were much
higher than normal SGC7901and BGC823gastric carcinoma cells(P<0.05).
Expression of E-cadherin were no difference between PDGF-B
overexpression and normal SGC7901and BGC823gastric carcinoma
cells(P>0.05).
Conclusions: Overexpression of PDGF-B increases the growth andinvasion of gastric carcinoma cells so as to promote tumor metastasis.Activation of PI3K/AKT signal pathway by PDGF-B autocrine might leadthe increases of the growth and invasion of gastric carcinoma cells.
PART Ⅳ THE RELATIONSHIP BETWEEN PDGF-BSIGNALING, EPITHELIAL–MESENCHYMALTRANSIYION AND METASTASIS OF GASTRICCARCINOMA AND ITS MECHANISM
Objective: To approach the relationships between PDGF-B signaling，epithelial–mesenchymal transition(EMT) and metastasis of gastriccarcinoma and to investigate the mechanism of PDGF-B signaling inmetastasis of gastric carcinoma.
Methods: Expressions of AKT-1, ERK-1, E-cadherin and N-cadherinprotein in SGC7901and BGC823gastric carcinoma cells before and afterPDGF-B transfection were detected by western-blot. Cell coculture wasused for activation of PDGF-B signaling. RT-PCR was used for detection ofmRNA of AKT-1, ERK-1, E-cadherin and N-cadherin in coculture PDGF-Boverexpression SGC7901and BGC823gastric carcinoma cells and incoculture normal SGC7901and BGC823gastric carcinoma cells, alsowestern-blot was used for detection of protein of AKT-1, ERK-1, E-cadherinand N-cadherin in coculture PDGF-B overexpression SGC7901andBGC823gastric carcinoma cells and in coculture normal SGC7901andBGC823gastric carcinoma cells.
Results: There were no differences between expressions of E-cadherin,N-cadherin and ERK-1protein in SGC7901and BGC823gastric carcinomacells before and after PDGF-B overexpression(P>0.05); the expressions ofAKT-1protein in PDGF-B overexpression SGC7901and BGC823gastriccarcinoma cells were much higher than those of in normal SGC7901andBGC823gastric carcinoma cells(P<0.05). The expressions of mRNA andprotein of AKT-1and ERK-1in cocultured PDGF-B overexpressionSGC7901and BGC823gastric carcinoma cells were much higher than thoseof in cocultured normal SGC7901and BGC823gastric carcinomacells(P<0.05). Expressions of mRNA and protein of EMT-related E-cadherinin coculture PDGF-B overexpression SGC7901and BGC823gastric carcinoma cells were much lower than those of in cocultured normalSGC7901and BGC823gastric carcinoma cells(P<0.05), and expressions ofmRNA and protein of ETM-related N-cadherin in cocultured PDGF-Boverexpression SGC7901and BGC823gastric carcinoma cells were muchhigher than those of in cocultured normal SGC7901and BGC823gastriccarcinoma cells(P<0.05). There were no differences between expressions ofAKT-1, ERK-1, E-cadherin and N-cadherin protein in normal SGC7901and BGC823gastric carcinoma cells before and after coculture(P>0.05).There were no differences between expressions of AKT-1protein inPDGF-B overexpression SGC7901and BGC823gastric carcinoma cellsbefore and after coculture(P>0.05); there were apparently differencesbetween expressions E-cadherin, N-cadherin and ERK-1protein inPDGF-B overexpression SGC7901and BGC823gastric carcinoma cellsbefore and after coculture(P<0.05).
Conclusions: PDGF-B signaling might not induce EMT in gastriccarcinoma cells through autocrine. PDGF-B signaling might induce EMT ingastric carcinoma cells through paracrine and joint action with tumorstromal cells so as to promote metastasis of gastric carcinoma. BothMAPK/ERK signal pathway(a downstream targets of PDGF signalingpathway) and tumor microenvironment might participate in the process ofEMT. Consequently, PDGF-B signaling might be an effective therapy targetfor gastric carcinoma.
引文
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