【作者】 李振华；

【导师】 高忠礼；

【作者基本信息】 吉林大学 ， 外科学， 2008， 博士

【摘要】 本研究以体外培养的人成骨肉瘤细胞系——U2OS及MG63细胞为对象,利用RNAi技术沉默细胞中人LIMK1基因,观察其对人成骨肉瘤细胞生物学行为的影响及作用机制。通过检测人成骨肉瘤细胞中LIMK1基因及蛋白表达发现,LIMK1在人成骨肉瘤细胞系MG63及U2OS细胞中高表达。本研究成功构建了siRNA真核表达质粒(pSUPER-LIMK1),瞬时转染显示RNA干涉LIMK1基因表达后,明显沉默LIMK1在人成骨肉瘤细胞中的基因及蛋白表达。在insulin促进细胞增殖的实验中,LIMK1信号通路激活,转染pSUPER-LIMK1质粒,使LIMK1基因沉默后,明显减弱insulin促进细胞增殖的作用。1,25(OH)2D3作为肿瘤的分化诱导剂,对人成骨肉瘤细胞主要通过促进骨钙素基因表达,改变细胞形态学、抑制肿瘤细胞增殖、改变肿瘤细胞周期变化发挥重要的作用。流式细胞仪检测LIMK1基因沉默后,人成骨肉瘤细胞停滞在S期,明显抑制了细胞的增殖,说明在1,25(OH)2D3促进人成骨肉瘤细胞分化中,LIMK1信号通路参与其中,发挥重要的调控作用。另外转染pSUPER-LIMK1质粒后,明显抑制了人成骨肉瘤细胞在细胞外基质的迁移和侵袭能力。本研究为以LIMK1为靶点的肿瘤发生机制和肿瘤治疗学研究提供了新的理论基础。更多还原

【Abstract】 Oseteosarcoma is a kind of common primary malignant neoplasms of bone. Most Oseteosarcoma attacks the adolescent, and causes great harm and high mortality. At the outset the disease is latent and in high-degree malignancy. Its early clinical symptoms may be confused with trauma of bone and pulmonary metastasis may occur in the early stage of the disease.At present, the main treatment of osteosarcoma is still surgical treatment. Yet some patients encounter treatment failure because of tumor recurrence and metastasis. Along with the development of experimentations on the molecular level and clinical research into tumor growth and invasion, a novel target was provided for gene therapy of osteosarcoma, which proves a broad prospect for research.LIM kinase, as a member of protein family, is a regulatory factor of cytoskeleton dynamics. LIM kinase has extensive biological effects and its major role is to participate in cellular cytoskeletal rearrangement. In recent years, the important role of LIMK1 played in the pathogenesis has drawn extensive attent ion. Research shows the overexpression of LIMK1 in prostate tumor and prostatic cancer cell lines and the expression of high concentration phosphated cofilin in prostate carcinoma metastasis cell. Inhibition of LIMK1 may arrest cell cycle at G/M stage and change cell proliferation and cell morphology, thus to contain the invasiveness of prostate carcinoma metastasis cell. The research also shows that the chromosomal gene site of prostate carcinoma metastasis is 7ql1.2, while that of human LIMK1 is 7ql 1.23 too, which suggests the correlation between them. It may be concluded that LIMK1 has an important regulatory effect on the division and invasiveness of tumor cells and it is probably one of the key moleculars which cause the invasiveness and metastasis of tumor cells. But there is still no related report of research on the correlation between LIMK1 and osteosarcoma.RNAi is a kind of sequence-specific and post-transcriptional gene silencing mechanism which is dsRNA triggered and which results in the degradation of sequence homology mRNA . At present, RNAi has been widely used in the study of genome function, anti-virus and anti-tumor.Insulin is a kind of hormone with varied biological effects. Different concentrations of insulin stimulates human osteosarcoma cells, and we found insulin in 500 ng / ml promoted cell proliferation obviously and in 5 ug / ml the promotion is the obviousest. 1 ug / ml insulin in different time points in human osteosarcoma cells showed that insulin’s role on cell proliferation is time-dependent. PD98059 and LY294002 are signal pathway inhibitors of MAPK and PI3K respectively. The two working together with insulin cells showed LY294002 significantly inhibiting insulin on the human osteosarcoma cell proliferation, while effects of PD98059 insignificant. It can be concluded that insulin promotes human osteosarcoma cell proliferation through the activation of PI3K signal pathway. By RT-PCR gene expression of LIMK1, at the transcriptional level LIMK1 did not change significantly, but Western Blot found cofilin expression and P-cofilin expression. When it works for 24 hours, P-cofilin expression heightens, and compared with the control group, it was statistically significant, suggesting that the promotion of insulin in the human osteosarcoma cell proliferation process initiated cofilin-associated signal transduction pathway. LIMK1 is the cofilin phosphatase, therefore the level of P-cofilin, in a sense, can note the activity of LIMK1. Further LIMK1 protein detection showed high LIMK1 expression, and proved that through the activation of LIMK1 signal transduction pathway and phosphorylation of cofilin, insulin tempted human osteosarcoma cell proliferation. After transfecting plasmid pSUPER -LIMK1 to human osteosarcoma to silence LIMK1 gene, insulin’s promotion of cell proliferation significantly weakened, suggesting that the activation of LIMK1 signal transduction pathway is important in insulin promoting human osteosarcoma cell proliferation.1,25 (OH) 2D3 plays an important role in inhibiting tumor cell proliferation and promoting tumor cell differentiation . But the message transmission mechanism of 1,25 (OH) 2D3 acting on tumor cell is still unknown. This experiment chose human osteosarcoma cell line to study, through CCK-8 assay to examine human osteosarcoma cell after stimulated by 10-7 mol / L 1,25 (OH) 2D3. Results from the experiment showed that compared with that of the control group, human osteosarcoma cell growth was significantly inhibited after stimulated by 10-7 mol / L 1,25 (OH) 2D3. By Westernblot assay we found the P-cofilin’s level was significantly increased after stimulated by 10-7 mol / L 1,25 (OH) 2D3 for 24 hours. But with the extension of time the P-cofilin level gradually drop. The inhibition of cell proliferation significantly weakened after the plasmid pSUPER -LIMK1 was transfected to human osteosarcoma cell, suggesting that LIMK1 signal pathway was activated in 1,25 (OH) 2D3 inhibiting human osteosarcoma cell proliferation.By RT-PCR, osteocalcin showed low expression in human osteosarcoma. Osteocalcin gene expression of human osteosarcoma cell stimulated by 10-8 mol / L 1,25 (OH) 2D3 was significantly increased and higher than that of stimulated by other concentration of 1,25 (OH) 2D3. Results showed that the best concentration of 1,25(OH)2D3 to promote differentiation of human osteosarcoma is 10-8 mol / L.We observed the ultra-microstructural changes of human osteosarcoma cells stimulated by 10-8 mol / L1, 25 (OH) 2D3 under TEM ,the results showed that along with the 1,25 (OH) 2D3 stimulating time increasing, cell nuclear shape became ruler, smaller and rounder, and cell surface microvilli disappeared, lysosome increased, suggesting that cells transformate from the naive poorly differentiated to maturity.We assessed cell cycle by FCM and found that cellular proportion in G2/M and S-phase was higher than other phase in the transfective non-carrier comparison group, consistent with that of human osteosarcoma cell depolarization characteristics. While in the experimental groups stimulated by 10-8 mol / L 1,25 (OH) 2D3, cellular proportion in S-phase(DNA synthetic phase) decreased significantly, most cells growth were arrestted at the G0/G1 phase ( resting stage) , suggesting that 1,25 (OH) 2D3 has the function of promoting differentiation obviously. Most cells were arrestted in S-phase in pSUPER- LIMK1 transfected group, after stimulated by 10-8 mol / L 1,25 (OH) 2D3, cellular proportion in S-phase decreased significantly too.To sum up, as the differentiation inducer of tumor, 1,25 (OH) 2D3 played an important role in changing cell morphology, inhibiting proliferation of tumor cell and changing tumor cell cycle mainly by improving the osteocalcin gene expression. LIMK1 plays an important role in the human osteosarcoma cell differentiation. After LIMK1 gene silencing, human osteosarcoma cells proliferation were significantly inhibited, the cell cycle was arrested in S-phase. At the same time in the 1,25 (OH) 2D3’s inducing differentiation process, the involvement of LIMK1 signal pathway played an important role in the regulation.Cell migration depends on actin cytoskeleton dynamic regulation. Cofilin mediates lamellipodium extension and polarizates cell migration through accelerating actin filament on the verge of cell migration. In the early stage of cell responses, LIMK1 can stimulate the cell-foot formation, therefore it is necessary in cell migration. Related literature shows that inhibition of LIMK1 can change cell morphology and curb the invasiveness of prostate carcinoma metastasis cell [5, 53]. It may be concluded that LIMK1 has an important regulatory effect on the division and invasiveness of tumor cells and it is probably one of the key moleculars which cause the invasiveness and metastasis of tumor cells.This study was to observe the impact of LIMK1 gene silencing after transfecting plasmid pSUPER-LIMK1 to human osteosarcoma cells vitro movement and migration through Boydern Chamber assay.Through the study, we found that human osteosarcoma cells movement in the extracellular matrix was significantly inhibittd after transfected plasmid pSUPER-LIMK1. Boydern Chamber assay showed that cell invasion significantly decreased after transfected plasmid pSUPER-LIMK1, and compared with the control group it was significantly different. The impact of LIMK1 on human osteosarcoma cells vitro movement and migration provides new drug targets for further gene therapy of osteosarcoma.更多还原