本文關(guān)鍵詞：1.帕米磷酸鈉對成骨不全成骨細(xì)胞和成纖維細(xì)胞增殖分化的研究 2.成骨不全成纖維細(xì)胞永生化細(xì)胞系的建立　出處：《濟(jì)南大學(xué)》2011年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 帕米磷酸鈉 成骨不全癥 成骨細(xì)胞 成纖維細(xì)胞 細(xì)胞增殖 ALP活性 永生化 猿猴病毒40大T抗原 成骨不全 成纖維細(xì)胞

【摘要】：背景與目的成骨不全(Osteogenisis Imperfecta)是一種由于間充質(zhì)組織發(fā)育不全、膠原形成障礙而造成的先天性遺傳性疾病,又稱脆骨癥(Fragililis ossium)、原發(fā)性骨脆癥(Idiopathic osteopsathyrosis)及骨膜發(fā)育不良(Periosteal dysplasia)。目前針對成骨不全的治療主要采用手術(shù)矯形后的髓內(nèi)針固定術(shù)及術(shù)后雙磷酸鹽藥物的輸液治療。 雙磷酸鹽類藥物可降低骨轉(zhuǎn)換,抑制骨吸收,臨床上常被用于治療一些骨吸收亢進(jìn)疾病,如絕經(jīng)后骨質(zhì)疏松癥、Puget’s病等。其主要作用機(jī)制是通過抑制破骨細(xì)胞內(nèi)甲羥戊酸通路的關(guān)鍵酶使胞內(nèi)的小G蛋白無法異戊二烯化,從而影響破骨細(xì)胞(osteoclast,OC)的生長和分化,促其凋亡。但近幾年的研究發(fā)現(xiàn),雙磷酸鹽可通過對成骨細(xì)胞的作用來間接抑制破骨細(xì)胞,而且對間充質(zhì)干細(xì)胞(bone marrow stormal cells, BMSCs)和成骨細(xì)胞(osteoblast,OB)的增殖和分化也有一定的影響。關(guān)于雙磷酸鹽對成纖維細(xì)胞(fibroblast,FB)的作用探討主要集中在雙磷酸鹽引起的頜骨壞死疾病(BRONJ)中,研究者發(fā)現(xiàn)采用不同劑量的唑來膦酸處理成纖維細(xì)胞,其中高于10μM濃度的唑來磷酸鹽即可引起成纖維細(xì)胞的程序性死亡,這種死亡機(jī)制部分是由胞內(nèi)的甲羥戊酸途徑介導(dǎo)的,而較低劑量的唑來磷酸鹽則促進(jìn)細(xì)胞增殖及IL-6,IL-8等細(xì)胞因子的表達(dá)。近幾年由于成骨不全癥骨質(zhì)疏松的臨床病理表現(xiàn),雙磷酸鹽類藥物被引入了該病的術(shù)后治療中。其中帕米磷酸鈉是主流的針對成骨不全癥的治療藥物。 本實(shí)驗(yàn)用不同濃度的帕米磷酸鈉作用于成骨不全患者成骨細(xì)胞和成纖維細(xì)胞,研究藥物對患者細(xì)胞增殖分化的影響。 方法: 1.收集成骨不全(OI)患者和對照組先天性髖脫位(dislocation of the hip,DDH)患者的股骨松質(zhì)骨和大腿皮膚組織,消化處理,收集細(xì)胞,進(jìn)行原代培養(yǎng)及鑒定。每一實(shí)驗(yàn)組及其對照組含有三例樣本。 2.采用10-3M～10-10M等8個(gè)帕米磷酸鈉藥物梯度處理實(shí)驗(yàn)組和對照組原代成骨細(xì)胞和成纖維細(xì)胞48h、72h。采用四甲基偶氮唑鹽(MTT)法檢測藥物對細(xì)胞增殖的影響。采用堿性磷酸酶(ALP)活性測定法(ELISA)檢測對成骨不全癥成骨細(xì)胞和成纖維細(xì)胞成骨分化的影響。 結(jié)果: 1.原代培養(yǎng)成骨不全癥和髖關(guān)節(jié)脫位患者成骨細(xì)胞和成纖維細(xì)胞,細(xì)胞生長狀態(tài)穩(wěn)定,可持續(xù)傳代。經(jīng)BCIP/NBT堿性磷酸酶顯色試劑盒染色鑒定成骨細(xì)胞堿性磷酸酶高表達(dá),成纖維細(xì)胞低表達(dá)。 2.藥物作用48h和72h均有利于細(xì)胞增殖,其中72h作用效果較明顯(P0.01)。10-3M,10-4M濃度帕米磷酸鈉明顯抑制兩組細(xì)胞增殖(P0.01);10-5M～10-10M濃度的帕米磷酸鈉對兩組細(xì)胞增殖有促進(jìn)作用,其中實(shí)驗(yàn)組成骨細(xì)胞和成纖維細(xì)胞最佳增殖濃度分別為10-8M和10-6M(P0.01),對應(yīng)增殖率可分別達(dá)到43%和39%。對照組成骨細(xì)胞和成纖維細(xì)胞的最佳增殖濃度分別為10-7M和10-5M(P0.01),對應(yīng)增殖率為37%和28%。 3.藥物濃度在10-5M～10-9M時(shí)都促進(jìn)細(xì)胞ALP表達(dá),且ALP活性變化與細(xì)胞增殖相一致。其中實(shí)驗(yàn)組成骨細(xì)胞和成纖維細(xì)胞最高ALP活性分別為102U/L和32U/L。 結(jié)論: 帕米磷酸鈉促進(jìn)實(shí)驗(yàn)組和對照組成骨細(xì)胞及成纖維細(xì)胞增殖能力和堿性磷酸酶的表達(dá),但是對于實(shí)驗(yàn)組細(xì)胞的作用更明顯。針對成骨不全病人適當(dāng)?shù)慕o藥方案有利于患者骨質(zhì)增強(qiáng)。 背景與目的:細(xì)胞原代培養(yǎng)費(fèi)事費(fèi)力,而且細(xì)胞傳代次數(shù)有限,在體外長期培養(yǎng)是細(xì)胞生理功能容易發(fā)生變異,造成實(shí)驗(yàn)結(jié)果因細(xì)胞代數(shù)的不同而難以比較。建立永生化的細(xì)胞系可以為細(xì)胞水平的研究提供一個(gè)有效的試驗(yàn)平臺,保證實(shí)驗(yàn)結(jié)果的一致性。本實(shí)驗(yàn)利用SV40T能促進(jìn)細(xì)胞永生化的功能,構(gòu)建含有SV40T基因的真核表達(dá)載體并轉(zhuǎn)染成骨不全成纖維細(xì)胞構(gòu)建穩(wěn)定細(xì)胞系,為成骨不全細(xì)胞水平實(shí)驗(yàn)提供穩(wěn)定細(xì)胞平臺。 方法: 1. PCI-neo-SV40T重組載體的構(gòu)建:限制性內(nèi)切酶XhoI和SalI雙酶切PAAV-SV40T,PCI-neo-hTERT得PCI-neo載體片段和SV40T基因片段。兩片段經(jīng)連接后轉(zhuǎn)化DH5α宿主菌,重組載體PCI-neo-SV40T經(jīng)PCR及酶切驗(yàn)證。 2. PCI-neo-SV40T重組質(zhì)粒通過脂質(zhì)體轉(zhuǎn)染經(jīng)鑒定的成骨不全原代成纖維細(xì)胞。 結(jié)果: 1.重組載體PCI-neo-SV40T經(jīng)XhoI與SalI雙酶切以及PCR驗(yàn)證正確。 2.重組質(zhì)粒PCI-neo-SV40T轉(zhuǎn)染原代成骨不全成纖維細(xì)胞后初期細(xì)胞生長穩(wěn)定,后逐漸死亡。 結(jié)論: 經(jīng)雙酶切和PCR驗(yàn)證,真核表達(dá)載體PCI-neo-SV40T構(gòu)建成功,為進(jìn)一步建立成骨不全成纖維細(xì)胞永生化細(xì)胞系奠定基礎(chǔ)
[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

【學(xué)位授予單位】：濟(jì)南大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R329

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本文編號：1431338