殼聚糖修飾的羥基磷灰石納米粒載體介導BMP-2轉(zhuǎn)染成骨細胞及對其增殖、分化的影響
發(fā)布時間：2018-11-08 20:17
【摘要】：目的：通過用殼聚糖（chitosan,CS）修飾的羥基磷灰石(hydroxyapatite, HA)納米粒載體攜帶骨形態(tài)發(fā)生蛋白2（Bone morphogenetic protein2,BMP2）基因轉(zhuǎn)染成骨細胞,研究CS-HA對成骨細胞的毒性反應、轉(zhuǎn)染后成骨細胞內(nèi)BMP-2基因表達情況及對細胞增殖與分化的影響，為基因修飾的成骨細胞的臨床研究奠定基礎。 方法：復蘇成骨細胞株并進行傳代培養(yǎng)；用殼聚糖對羥基磷灰石納米粒進行表面修飾；采用MTT法檢測HA-CS納米粒載體對成骨細胞的毒性反應；用HA-CS納米粒載體轉(zhuǎn)染技術,將BMP-2目的基因?qū)氤晒羌毎?評估轉(zhuǎn)染效率并采用RT-PCR法鑒定BMP-2的表達;同時檢測細胞增殖情況及堿性磷酸酶（alkaline phoaphatase，ALP）活性。 結(jié)果：MTT結(jié)果提示HA-CS納米�；鞈乙簼舛仍�500μg/ml以下對成骨細胞生長的影響與對照組比較差異無統(tǒng)計學意義；其轉(zhuǎn)染效率雖不及同等條件下的脂質(zhì)體但優(yōu)于沒經(jīng)修飾的HA; RT-PCR結(jié)果顯示轉(zhuǎn)染后同對照組及空質(zhì)粒組比較成骨細胞內(nèi)BMP-2穩(wěn)定表達并表達增強;同時也促進了成骨細胞的增殖及增加了ALP的活性。 結(jié)論：HA-CS納米粒在一定濃度下對成骨細胞無毒性作用,作為輸送載體可以安全地將目的基因BMP-2導入成骨細胞并穩(wěn)定表達，，同時促進了成骨細胞的增殖與分化。
[Abstract]:Aim: to study the toxic effect of chitosan,CS on osteoblasts by transfection of hydroxyapatite (hydroxyapatite, HA) nanoparticles carrying bone morphogenetic protein-2 (Bone morphogenetic protein2,BMP2 gene into osteoblasts. The expression of BMP-2 gene in osteoblasts after transfection and its effect on cell proliferation and differentiation lay a foundation for the clinical study of genetically modified osteoblasts. Methods: resuscitation osteoblasts were subcultured, hydroxyapatite nanoparticles were modified with chitosan, and the toxicity of HA-CS nanoparticles to osteoblasts was detected by MTT method. The BMP-2 target gene was transfected into osteoblasts by HA-CS nanoparticles transfection technique. The transfection efficiency was evaluated and the expression of BMP-2 was identified by RT-PCR method. The proliferation and alkaline phosphatase (alkaline phoaphatase,ALP) activity were also detected. Results: the results of MTT showed that there was no significant difference in the effect of the concentration of HA-CS nanoparticles suspension below 500 渭 g/ml on the growth of osteoblasts compared with the control group. The transfection efficiency was lower than that of liposomes under the same conditions, but better than that of unmodified HA; RT-PCR. The results showed that the stable expression and expression of BMP-2 in osteoblasts after transfection were higher than those in control group and empty plasmid group. It also promoted the proliferation of osteoblasts and increased the activity of ALP. Conclusion: HA-CS nanoparticles have no toxic effect on osteoblasts at a certain concentration. As a carrier, the target gene BMP-2 can be safely transferred into osteoblasts and expressed stably, and the proliferation and differentiation of osteoblasts can be promoted at the same time.
【學位授予單位】：佳木斯大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R318.08
[Abstract]:Aim: to study the toxic effect of chitosan,CS on osteoblasts by transfection of hydroxyapatite (hydroxyapatite, HA) nanoparticles carrying bone morphogenetic protein-2 (Bone morphogenetic protein2,BMP2 gene into osteoblasts. The expression of BMP-2 gene in osteoblasts after transfection and its effect on cell proliferation and differentiation lay a foundation for the clinical study of genetically modified osteoblasts. Methods: resuscitation osteoblasts were subcultured, hydroxyapatite nanoparticles were modified with chitosan, and the toxicity of HA-CS nanoparticles to osteoblasts was detected by MTT method. The BMP-2 target gene was transfected into osteoblasts by HA-CS nanoparticles transfection technique. The transfection efficiency was evaluated and the expression of BMP-2 was identified by RT-PCR method. The proliferation and alkaline phosphatase (alkaline phoaphatase,ALP) activity were also detected. Results: the results of MTT showed that there was no significant difference in the effect of the concentration of HA-CS nanoparticles suspension below 500 渭 g/ml on the growth of osteoblasts compared with the control group. The transfection efficiency was lower than that of liposomes under the same conditions, but better than that of unmodified HA; RT-PCR. The results showed that the stable expression and expression of BMP-2 in osteoblasts after transfection were higher than those in control group and empty plasmid group. It also promoted the proliferation of osteoblasts and increased the activity of ALP. Conclusion: HA-CS nanoparticles have no toxic effect on osteoblasts at a certain concentration. As a carrier, the target gene BMP-2 can be safely transferred into osteoblasts and expressed stably, and the proliferation and differentiation of osteoblasts can be promoted at the same time.
【學位授予單位】：佳木斯大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R318.08
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