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Experimental Study That Has-mir-129 Regulates the Biological Characters of Renal Cell Carcinoma SN12-PM6

Author: ZengSiPing
Tutor: XiaoYaJun
School: Huazhong University of Science and Technology
Course: Surgery
Keywords: miRNAs (microRNAs) Transfection Renal cell carcinoma Lung metastasis Model Orthotopic Tumor of urology
CLC: R737.11
Type: PhD thesis
Year: 2009
Downloads: 184
Quote: 1
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Abstract


Renal cancers are a kind of common renal malignant tumors and originfrom renal tubular epithelial cells, which account for 2-3% of all adultmalignant tumors and 85-90% of primary malignant renal tumors. Theincidence is only the second to bladder cancers. Previous reports indicate that30% of renal cancers have combined with distant metastasis when patientswere diagnosed as renal malignant tumors and there are a large proportion ofpatients with lung metastases. In recent years, advances in imaging technologyhave taken place, especially the popularity of B-ultrasonography and theupgrade of CT have improved the diagnosis and treatment of renal cellcarcinomas, which have resulted in the increase of renal cell carcinomas. Foryears, researches confirm that chemotherapy, radiotherapy, hormone treatmenthave bad effects on renal cell carcinomas. Immunotherapy has currently beenconsidered the most valuable treatment, but the efficient is less than 35%. The treatment of renal cell carcinomas is a lack of effective methods. Studieshave indicated that miRNAs are likely to be a new potential way for tumors.MicroRNAs (miRNAs) are a widely distributed class of endogenous smallnon-coding RNAs with 22 nucleotides in length. MicroRNAs control targetgene expression or translation by target mRNAs. MicroRNAs with highlyconserved sequences exist in a wide range including animals, plants andviruses. They have an important role in cell proliferation, differentiation andapoptosis, and regulate eukaryotic gene expression. There are two aspects thatmiRNAs regulate tumors. At first, miRNAs can control proto-oncogene andthus inhibit tumors. Secondly, they can also regulate tumor suppressor genesand play a role of ongenes. The current studies find that miRNAs havedifferent levels in different tumor tissues and many miRNAs in tumor tissuesare significantly lower than normal tissues, but the expression of miRNAsincreases in some tumor tissues, which indicates that the abnormal expressionof miRNAs may be closely related to the genesis of tumors.We have transfected renal cancer cell lines (SN12-PM6) with has-mir-129by the liposome-mediated method and observed the impact on renal cancercell lines in vitro. The model of renal cancer with lung metastasis has beenestablished. And we have observed that has-mir-129 impacted orthotopicmouse model of human renal cell carcinoma with pulmonary metastasis. The study has been divided into three parts and the results are as follows.Part One. The study that has-mir-129 has impacted on the growth of renalcancer cell lines (SN12-PM6) after SN12-PM6 was transfected withhas-mir-129The part is the major role that has-mir-129 impacts the growth,the invasion ability, reproductive activity and the cell cycle of renal carcinomacell lines (SN12-PM6). After plasmid amplification and extraction, theplasmid of has-mir-129(GFP) can be obtained. SN12-PM6 cells weretransfected with has-mir-129(GFP) and screened by flow cytometry.SN12-PM6 cells with green fluorescent were cultured. The cell growth curvewas detected by MTT method. The homogeneous adhesion capacity, theinvasion and migration capacity (Boyden chamber), the proliferative capacity(Brdu method) has been detected. The results indicate that the growth ofSN12-PM6 cells was inhibited, the homogeneous adhesion, invasion andmigration capacity decreased, (P<0.05), but DNA replication capacity was nosignificant difference in S phase after SN12-PM6 cells were transfected withmiR-129.Part Two. Establishment of orthotopic mouse model of human renal cell carcinoma with pulmonary metastasisThe part is the role to construct orthotopic mouse model ofhuman renal cell carcinoma with pulmonary metastasis and to study the firsttime of pulmonary metastasis in nude mice. SN12-PM6 was transplanted intothe right kidey of nude mice. Tumorigenicity and metastasis were evaluatedsubsequently. All tumor tissues were fixed in 10% formalin and embedded inparaffin routinely. Paraffin-embedded tissues were stained with hematoxylinand eosin (HE) for routine histological examination. The results indicate thatthe incidences of tumorigenicity were 100% (39/39). In the fifth weekpulmonary metastasis were observed and the incidences of metastasis were50% (4/8). In the subsequent 3 weeks the incidences of metastasis were 75%(3/4), 100% (8/8) and 100% (7/7), respectively. The metastasis of Intestinaltracts, livers, spleens was not observed obviously.Part Three. Study that has-mir-129 impacted orthotopic mouse model ofhuman renal cell carcinoma with pulmonary metastasisThe part is the role to construct orthotopic mouse model ofhuman renal cell carcinoma with pulmonary metastasis and to study theimpact of has-mir-129(GFP) on renal transplantation tumor and lungmetastases. Human renal cancer cell line SN12-PM6 set up the animal model as the control group, and Human renal cancer cell lines SN12-PM6 beinginfected the lentivirus with has-mir-129(GFP) were cultured in order to set upthe animal model as the experimental group. After seven weekstumorigenicity and metastasis were evaluated subsequently. In the two groups,the percentage of tumorigenicity and metastasis was calculated; at the sametime, the weight of tumor was also taken into account. All tumor tissues werefixed in 10% formalin and embedded in paraffin routinely. Paraffin-embeddedtissues were stained with hematoxylin and eosin (HE) for routine histologicalexamination. Results: In the control group, the percentage of tumorigenicitywas 100% (8 / 8), the percentage of metastasis was 100% (8 / 8) and theaverage tumor weight was (0.7128±0.1474) g. But in the experimental thepercentage of tumorigenicity was 100% (8 / 8), the percentage of metastasiswas 62.5% (5 / 8) and the average tumor weight was (0.4815±0.1112) g.Statistics show that the percentage of tumorigenicity was no significantdifference between the two groups, but the average tumor weight and thepercentage of metastasis was significantly different between the two groups, P<0.05.Review articles. MicroRNAs and Advances of Urinary TumorsMicroRNAs(miRNAs) are universally small non-coding RNA that are widely reported in plants, animals, and even viruses. It has beendemonstrated that miRNAs are associated that development and prognosis oftumors, and miRNAs play an important role of proliferation, differentiationand apoptosis of tumor. In this review article, we will discuss the progress onthe study of miRNAs and urinary tumors.Conclusion①The growth of SN12-PM6 cells was inhibited, the homogeneous adhesion,invasion and migration capacity decreased after SN12-PM6 cells weretransfected with has-mir- 129 (P<0.05).②Has-mir-129 can inhibit the growth of SN12-PM6 cells, but DNAreplication capacity was no significant difference in S phase after SN12-PM6cells were transfected with has-mir-129(GFP), and its mechanism may berelated to inhibition of the G1 phase.③We have successfully constructed orthotopic mouse model of human renalcell carcinoma with pulmonary metastasis and provided an ideal researchmodel of renal cell carcinoma, especially lung metastasis of renal cellcarcinoma.④Five weeks later pulmonary metastasis were observed and the incidencesof metastasis were 50%. The metastasis of Intestinal tracts, livers, spleens was not observed obviously.⑤We have successfully constructed orthotopic mouse model of human renalcell carcinoma with pulmonary metastasis and found that has-mir-129 caninhibit tumor growth and pulmonary metastasis of orthotopic mouse model ofhuman renal cell carcinoma with pulmonary metastasis.

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CLC: > Medicine, health > Oncology > Genitourinary tumors > Urinary tumors > Kidney,renal pelvis tumor
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