【作者】 赵华；

【导师】 赵红宇；

【作者基本信息】 郑州大学 ， 口腔临床医学， 2007， 硕士

【摘要】 目的观察Emdogain、辛伐他汀及两者联合应用对人牙周膜细胞增殖和分化能力的影响方法1、牙周膜组织的获得：12～25岁因正畸治疗需要拔除的恒牙，无全身疾病，实验所需牙齿无龋病、牙周病、根尖周病等，严格消毒后将拔出的牙齿放入无菌的PBS溶液中，2小时内刮取牙周膜组织培养牙周膜细胞。2、人牙周膜细胞培养：人牙周膜细胞在含15％小牛血清的DMEM培养液中进行培养，细胞接近融合成单层时，0.25％胰蛋白酶常规消化，在倒置相差显微镜下观察细胞分离、胞质回缩，70％细胞脱壁后，终止消化，收集消化所得的细胞待用。本实验所采用均为第4至第6代细胞。3、人牙周膜细胞鉴定：a)倒置相差显微镜形态学观察b)矿化结节染色(茜素红染色法)c)免疫组化鉴定4、观察不同浓度的Emdogain(200mg／L、100mg／L、50mg／L、25mg／L)对人牙周膜细胞增殖和分化能力的影响：a)四甲基偶氮唑盐微量酶反应比色法(MTT法)测定细胞增殖b) PNPP偶氮法检测细胞的碱性磷酸酶活性c)Ⅰ型胶原检测(羟脯氨酸含量测定)5、观察不同浓度的辛伐他汀(0.125μmol／L、0.25μmol／L、0.5μmol／L、1.0μmol／L)对人牙周膜细胞增殖和分化能力的影响：a) MTT法测定细胞增殖b) PNPP偶氮法检测细胞的碱性磷酸酶活性c)Ⅰ型胶原检测(羟脯氨酸含量测定) 6、观察浓度0.125μmol／L的辛伐他汀和浓度50mg／LEmdogain联合作用对人牙周膜细胞增殖和分化能力的影响：a) MTT法测定细胞增殖b) PNPP偶氮法检测细胞的碱性磷酸酶活性c)Ⅰ型胶原检测(羟脯氨酸含量测定)结果1、本实验所培养细胞形态正常，状态良好，性状稳定。此外，免疫组化染色角蛋白染色阴性，波形丝蛋白染色阳性，证明细胞是来源于外胚间充质的牙周膜细胞，可用于后续实验。2、不同浓度的Emdogain(200mg／L、100mg／L、50mg／L、25mg／L)对人牙周膜细胞增殖和分化能力的影响：a)细胞增殖能力测定：与对照组比较，25mg／L、50mg／L、100mg／L浓度Emdogain均可促进人牙周膜细胞增殖，对照组与实验组比较差异有统计学意义(P＜0.05)，但实验组组间比较差异无统计学意义，且本实验数据显示50mg／L作用较强。b)细胞碱性磷酸酶活性测定：与对照组比较，25mg／L、50mg／L、100mg／L浓度Emdogain可增加人牙周膜细胞的碱性磷酸酶活性，差异有统计学意义(P＜0.05)，但组间比较差异无统计学意义，且本实验数据显示50mg／L浓度作用较强，而200mg／L浓度组在实验期间不能提高人牙周膜细胞的碱性磷酸酶的活性。c)Ⅰ型胶原检测：25mg／L、50mg／L、100mg／L、200mg／L4个浓度Emdogain可增强人牙周膜细胞形成Ⅰ型胶原的能力(P＜0.05)，且本实验数据显示50mg／L浓度作用较强。3、不同浓度的辛伐他汀(0.125μmol／L、0.25μmol／L、0.5μmol／L、1.0μmol／L)对人牙周膜细胞增殖和分化能力的影响：a)细胞增殖能力测定：与对照组比较，各浓度组辛伐他汀抑制人牙周膜细胞增殖，对照组与实验组比较差异有统计学意义(P＜0.05)。b)细胞碱性磷酸酶活性测定：与对照组比较，0.125μmol／L、0.25μmol／L、0.5μmol／L浓度辛伐他汀可增加人牙周膜细胞的碱性磷酸酶活性，差异有统计学意义(P＜0.05)，但组间比较差异无统计学意义，且本实验数据显示0.125μmol／L浓度作用较强。c)Ⅰ型胶原检测：0.125μmol／L、0.25μmol／L、0.5μmol／L、1.0μmol／L4个浓度梯度辛伐他汀可增强人牙周膜细胞形成Ⅰ型胶原的能力，但组间比较差异无统计学意义，且本实验数据显示0.125μmol／L浓度作用较强。4、观察浓度0.125μmol／L的辛伐他汀和浓度50mg／L Emdogain联合作用对人牙周膜细胞增殖和分化能力的影响：a)细胞增殖能力测定：二者联合对人牙周膜细胞的增殖作用不明显。b)细胞碱性磷酸酶活性测定：对照组与实验组比较差异有统计学意义(P＜0.05)，二者联合对人牙周膜细胞的碱性磷酸酶活性有促进作用。c)Ⅰ型胶原检测：对照组与实验组比较差异有统计学意义(P＜0.05)，二者联合对人牙周膜细胞合成Ⅰ型胶原能力有促进作用。结论1、Emdogain可促进人牙周膜细胞增殖、碱性磷酸酶活性、合成Ⅰ型胶原能力增强；2、辛伐他汀抑制人牙周膜细胞的增殖，但可促进其碱性磷酸酶活性和合成Ⅰ型胶原能力的增强；3、两者联合应用对细胞的增殖影响甚微，可促进细胞的碱性磷酸酶活性和合成Ⅰ型胶原的能力：4、这为二者联合应用于心血管病、糖尿病患者的牙周病治疗提供了一定的参考价殖，但具体的用药方式、用药次序、用药时间及用药浓度配伍等问题还需要进一步的研究。更多还原

【Abstract】 ObjectsTo observe the effects of Emdqgain、Simvastatin and their combination on the proliferation and differentiation of human periodontal ligament cells(HPLCs)Methods1、Preparation of periodontal ligament tissue.Eight periodontally healthy and non-carious premolars extracted in the course of orthodontic treatment were collected from two patients with their informed consent. Then the teeth were putted into the sterile PBS solution, and the periodontal ligament tissue which attached to the mid-third of each root was extracted within two hours to culture the periodontal ligament cells.2、Preparation and culture of HPLCsHPLCs were cultured to confluence in a 75mm~2 culture flask with Dulbecco’s modified Eagle’s medium (DMEM)supplemented with 15% fetal bovine serum, 100 U:ml penicillin, 100 mg:ml streptomycin and 1 mg:ml amphotericin B (medium A). When HPLCs formed a confluent monolayer, cells were harvested with 0. 25% trypsin transferred to a 150mm~2 culture flask. HPLCs in cultures at the 4th、5th、6th passage were used in the following experiments.3、Indentification for human periodontal ligament cells ①Cells morphology on phase-contrast microscope.②Mineralization nodule staining(Alizarin Bordeaux staining method),③Indentification on immunohistochemistry4, To observe the effects of Emdogain(200、100、50、25mg/L )on the proliferation anddifferentiation of human periodontal ligament cells①To assay proliferation of HPLCs by MTT.②To assay ALP activity of HPLCs by PNPP.③Type- I collagen in HPLCs were detected by assaying the level of hydroxyproline.5. To observe the effects of Simvastatins(0.125μmol/L、0.25μmol/L、0.5μmol/L、1.0μmol/L)on the proliferation and differentiation of human periodontal ligament cells①To assay proliferation of HPLCs by MTT.②To assay ALP activity of HPLCs by PNPP.③Type-I collagen in HPLCs were detected by assaying the level of hydroxyproline.6、To observe the effects of the combination of 50mg/L Emdogain and 0.125μmol/L Simvastatins on the proliferation and differentiation of human periodontal ligament cells①To assay proliferation of HPLCs by MTT.②To assay ALP activity of HPLCs by PNPP.③Type- I collagen in HPLCs were detected by assaying the level of hydroxyproline.Results1、To observe the effects of Emdogain(200 mg/L、100 mg/L、50 mg/L、25mg/L)on the proliferation and differentiation of human periodontal ligament cells. Compared with control group, Emdogain(100 mg/L、50 mg/L、25mg/L)can increase the proliferation, the synethesis of Type- I collagen of HPLCs.Compared with control group ,Emdogain(100 mg/L、50 mg/L、25mg/L )can increase the ALP activity ,but 200mg/L Emdogain can not increase the ALP activity during the experiment. No difference was found among treated groups,and the effects of 50mg/L Emdogain was the highest(P<0.05) according to the datas of this experiment.2. The effects of Simvastatins(0.125μmol/L、0. 25μmol/L、0.5μmol/L、1.0μmol/L) on the proliferation and differentiation of human periodontal ligament cells. Compared with control group, Simvastatins(0.125μmol/L、0. 25μmol/L、0.5μmol/L、1.0μmol/L )can increase the ALP activity and the synethesis of Type- I collagen of HPLCs, no differences was found among treated groups ,and the value of 0.125μmol/L Simvastatins was highest.(P<0.05) according to this experiment;compared with control group, Simvastatins(0.125μmol/L、0. 25μmol/L、0.5μmol/L、1.0μmol/L )significantly inhibited the proliferation of HPLCs.3、To observe the effects of 50mg/L Emdogain and 0.125μmol/L Simvastatins on the proliferation and differentiation of human periodontal ligament cells.Compared with control group, treated groups can increase the ALP activity and the synethesis of Type- I collagen in HPLCs, but treated groups have little effects on the proliferation of HPLCs.Conclusions1、Emdogain can promote the proliferation、ALP activity、the synthesis of Collagen type I ;2、Simvastain have the same effects on the differentiation of HPLCs,but inhibit the proliferation of HPLCs.3、Their combination have little effects on the proliferation of HPLCs,but significantly stimulate the differentiation of HPLCs.4、All above may give the experiment proofs to the treatment of periodontal disease combinated with cardiovascular disease and diabetes,but their concrete ways、sequence、time and concentration of the medicince in clinical application are worthy of further research.更多还原