1.帕米磷酸钠对成骨不全成骨细胞和成纤维细胞增殖分化的研究 2.成骨不全成纤维细胞永生化细胞系的建立

发布时间：2018-01-16 03:22

本文关键词：1.帕米磷酸钠对成骨不全成骨细胞和成纤维细胞增殖分化的研究 2.成骨不全成纤维细胞永生化细胞系的建立　出处：《济南大学》2011年硕士论文　论文类型：学位论文

【摘要】：背景与目的成骨不全(Osteogenisis Imperfecta)是一种由于间充质组织发育不全、胶原形成障碍而造成的先天性遗传性疾病,又称脆骨症(Fragililis ossium)、原发性骨脆症(Idiopathic osteopsathyrosis)及骨膜发育不良(Periosteal dysplasia)。目前针对成骨不全的治疗主要采用手术矫形后的髓内针固定术及术后双磷酸盐药物的输液治疗。双磷酸盐类药物可降低骨转换,抑制骨吸收,临床上常被用于治疗一些骨吸收亢进疾病,如绝经后骨质疏松症、Puget’s病等。其主要作用机制是通过抑制破骨细胞内甲羟戊酸通路的关键酶使胞内的小G蛋白无法异戊二烯化,从而影响破骨细胞(osteoclast,OC)的生长和分化,促其凋亡。但近几年的研究发现,双磷酸盐可通过对成骨细胞的作用来间接抑制破骨细胞,而且对间充质干细胞(bone marrow stormal cells, BMSCs)和成骨细胞(osteoblast,OB)的增殖和分化也有一定的影响。关于双磷酸盐对成纤维细胞(fibroblast,FB)的作用探讨主要集中在双磷酸盐引起的颌骨坏死疾病(BRONJ)中,研究者发现采用不同剂量的唑来膦酸处理成纤维细胞,其中高于10μM浓度的唑来磷酸盐即可引起成纤维细胞的程序性死亡,这种死亡机制部分是由胞内的甲羟戊酸途径介导的,而较低剂量的唑来磷酸盐则促进细胞增殖及IL-6,IL-8等细胞因子的表达。近几年由于成骨不全症骨质疏松的临床病理表现,双磷酸盐类药物被引入了该病的术后治疗中。其中帕米磷酸钠是主流的针对成骨不全症的治疗药物。本实验用不同浓度的帕米磷酸钠作用于成骨不全患者成骨细胞和成纤维细胞,研究药物对患者细胞增殖分化的影响。方法: 1.收集成骨不全(OI)患者和对照组先天性髋脱位(dislocation of the hip,DDH)患者的股骨松质骨和大腿皮肤组织,消化处理,收集细胞,进行原代培养及鉴定。每一实验组及其对照组含有三例样本。 2.采用10-3M～10-10M等8个帕米磷酸钠药物梯度处理实验组和对照组原代成骨细胞和成纤维细胞48h、72h。采用四甲基偶氮唑盐(MTT)法检测药物对细胞增殖的影响。采用碱性磷酸酶(ALP)活性测定法(ELISA)检测对成骨不全症成骨细胞和成纤维细胞成骨分化的影响。结果: 1.原代培养成骨不全症和髋关节脱位患者成骨细胞和成纤维细胞,细胞生长状态稳定,可持续传代。经BCIP/NBT碱性磷酸酶显色试剂盒染色鉴定成骨细胞碱性磷酸酶高表达,成纤维细胞低表达。 2.药物作用48h和72h均有利于细胞增殖,其中72h作用效果较明显(P0.01)。10-3M,10-4M浓度帕米磷酸钠明显抑制两组细胞增殖(P0.01);10-5M～10-10M浓度的帕米磷酸钠对两组细胞增殖有促进作用,其中实验组成骨细胞和成纤维细胞最佳增殖浓度分别为10-8M和10-6M(P0.01),对应增殖率可分别达到43%和39%。对照组成骨细胞和成纤维细胞的最佳增殖浓度分别为10-7M和10-5M(P0.01),对应增殖率为37%和28%。 3.药物浓度在10-5M～10-9M时都促进细胞ALP表达,且ALP活性变化与细胞增殖相一致。其中实验组成骨细胞和成纤维细胞最高ALP活性分别为102U/L和32U/L。结论: 帕米磷酸钠促进实验组和对照组成骨细胞及成纤维细胞增殖能力和碱性磷酸酶的表达,但是对于实验组细胞的作用更明显。针对成骨不全病人适当的给药方案有利于患者骨质增强。背景与目的:细胞原代培养费事费力,而且细胞传代次数有限,在体外长期培养是细胞生理功能容易发生变异,造成实验结果因细胞代数的不同而难以比较。建立永生化的细胞系可以为细胞水平的研究提供一个有效的试验平台,保证实验结果的一致性。本实验利用SV40T能促进细胞永生化的功能,构建含有SV40T基因的真核表达载体并转染成骨不全成纤维细胞构建稳定细胞系,为成骨不全细胞水平实验提供稳定细胞平台。方法: 1. PCI-neo-SV40T重组载体的构建:限制性内切酶XhoI和SalI双酶切PAAV-SV40T,PCI-neo-hTERT得PCI-neo载体片段和SV40T基因片段。两片段经连接后转化DH5α宿主菌,重组载体PCI-neo-SV40T经PCR及酶切验证。 2. PCI-neo-SV40T重组质粒通过脂质体转染经鉴定的成骨不全原代成纤维细胞。结果: 1.重组载体PCI-neo-SV40T经XhoI与SalI双酶切以及PCR验证正确。 2.重组质粒PCI-neo-SV40T转染原代成骨不全成纤维细胞后初期细胞生长稳定,后逐渐死亡。结论: 经双酶切和PCR验证,真核表达载体PCI-neo-SV40T构建成功,为进一步建立成骨不全成纤维细胞永生化细胞系奠定基础
[Abstract]:Background and objective osteogenesis imperfecta (Osteogenisis Imperfecta) is a kind of mesenchymal tissue due to hypoplasia of disease, congenital disorders caused by collagen formation, also called osteopsathyrosis (Fragililis ossium), primary osteopsathyrosis (Idiopathic osteopsathyrosis) and periosteal dysplasia (Periosteal dysplasia) at present. The infusion of bisphosphonate therapy into bone insufficiency mainly by surgical correction after intramedullary nail fixation and postoperative.
Bisphosphonates can reduce bone turnover, inhibit bone resorption, clinically it is often used to treat some diseases such as bone resorption, postmenopausal osteoporosis, Puget 's disease. The main mechanism is the key enzyme inhibition of osteoclast in the mevalonate pathway of intracellular G protein is not small prenylation, thus affecting osteoclasts (osteoclast, OC) growth and differentiation, apoptosis. But recent researches have found that bisphosphonates through effects on osteoblast indirectly inhibit osteoclasts, and of mesenchymal stem cells (bone marrow stormal cells, and BMSCs) bone cells (osteoblast, OB) on the proliferation and differentiation also have certain effect. A bisphosphonate on fibroblast (fibroblast, FB) mainly focus on the discussion of the role of bisphosphonates in osteonecrosis of the jaw disease (BRONJ), researchers found that using different doses of The zoledronic acid treatment of fibroblasts, which is higher than the 10 M concentration of phosphate can induce programmed cell death into fibers, part of the death mechanism of the mevalonate pathway is mediated by intracellular conduction, and low dose of phosphate promotes cell proliferation and IL-6 expression of IL-8. Other cytokines. In recent years, due to clinical and pathological manifestations of osteogenesis imperfecta osteoporosis, bisphosphonates have been introduced into the disease after surgical treatment. Among them is the mainstream of pamidronate for treatment of osteogenesis imperfecta.
In this experiment the effects of different concentrations of pamidronate in patients with osteogenesis imperfecta, osteoblasts and fibroblasts, effects of drugs on cell proliferation and differentiation of patients.
Method:
1. collect femur cancellous bone and thigh skin tissue of patients with OI dislocation and control group of of the hip (DDH). Digestion and treatment, collecting cells, primary culture and identification. Each experimental group and its control group contain three samples.
Using 10-3M 2. ~ 10-10M 8 pamidronate drug gradient treatment experimental group and control group of primary osteoblasts and fibroblasts of 48h 72h., using four methyl thiazolyl tetrazolium (MTT) method was used to detect the effects of drugs on cell proliferation. The alkaline phosphatase (ALP) activity assay (ELISA) detection of OI osteoblasts and fibroblasts osteogenic differentiation.
Result:
1., primary osteoblasts and fibroblasts in patients with osteogenesis imperfecta and hip dislocation were stable and passable. The expression of alkaline phosphatase in osteoblasts was highly expressed by BCIP/NBT alkaline phosphatase staining kit, and fibroblasts were low expressed.
2. drugs 48h and 72h are conducive to cell proliferation, the 72h effect is obvious (P0.01).10-3M, 10-4M concentration of pamidronate significantly inhibited cell proliferation in two groups (P0.01); 10-5M ~ pamidronate 10-10M concentration has a promoting effect on the proliferation of two cells in the experimental group, the composition of bone cells and fibroblast proliferation best cell concentrations were 10-8M and 10-6M (P0.01), can reach 43% and 39%. respectively control the composition of bone cells and proliferation of fibroblasts into optimal concentration were 10-7M and 10-5M (P0.01), the proliferation rate corresponding to the corresponding proliferation rate was 37% and 28 respectively.
3., the drug concentration at 10-5M ~ 10-9M promoted ALP expression, and ALP activity was consistent with cell proliferation. The highest ALP activity of bone cells and fibroblasts was 102U/L and 32U/L..
Conclusion:
Pamidronate promote the experimental group and the control expression of composition and fibroblast proliferation and alkaline phosphatase in bone cells, but the cells in the experimental group had better effect on osteogenesis imperfecta patients. Appropriate dosage regimen is beneficial to patients with bone strengthening.
Background and objective: primary cell culture and time and energy consuming, the number of cells in long-term culture in vitro is limited, cell physiological function prone to mutation, the experimental results for cell algebra is different and difficult to compare. Provide an effective test platform for establishment of immortalized cell lines for cell level, ensure consistency the result of the experiment. This experiment using SV40T can promote cell immortalization, construct the eukaryotic expression vector of SV40T gene transfection and osteogenesis imperfecta fiber cells to construct stable cell lines, providing a stable cell platform for osteogenesis imperfecta cell level experiments.
Method:
1., the construction of PCI-neo-SV40T recombinant vector: restriction endonuclease XhoI and SalI double enzyme digestion PAAV-SV40T, PCI-neo-hTERT obtained PCI-neo carrier fragment and SV40T gene fragment. Two fragments were transformed into DH5 alpha host bacteria after linkage, and recombinant vector PCI-neo-SV40T was verified by PCR and enzyme digestion.
2. PCI-neo-SV40T recombinant plasmids were transfected through liposomes to identify osteoblast fibroblasts.
Result:
1. the recombinant vector PCI-neo-SV40T was verified by both XhoI and SalI double enzyme digestion and PCR.
2. recombinant plasmid PCI-neo-SV40T was transfected into primary osteogenesis imperfecta fibroblasts after initial cell growth is stable, gradually after death.
Conclusion:
After double enzyme digestion and PCR verification, the eukaryotic expression vector PCI-neo-SV40T was constructed successfully, which lays a foundation for the further establishment of osteogenesis imperfecta fibroblasts immortalized cell lines

【学位授予单位】：济南大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R329

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