發(fā)布時(shí)間：2018-01-17 02:03

  本文關(guān)鍵詞：脈沖電磁場對脫鈣骨基質(zhì)誘導(dǎo)人骨髓間充質(zhì)干細(xì)胞增殖與成骨分化的影響　出處：《第三軍醫(yī)大學(xué)》2010年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 脈沖電磁場 脫鈣骨基質(zhì) 人骨髓間充質(zhì)干細(xì)胞 增殖 成骨分化

【摘要】： 背景和目的:脫鈣骨基質(zhì)(Demineralized Bone Matrix, DBM)具有在體外促進(jìn)人骨髓間充質(zhì)干細(xì)胞(human Mesenchymal Stem Cells, hMSCs)增殖與成骨分化,在體內(nèi)促進(jìn)新骨形成的能力,在臨床組織工程中具有廣泛應(yīng)用價(jià)值。hMSCs具有良好的成骨細(xì)胞分化潛能和成骨活性,是骨組織工程種子細(xì)胞的最佳選擇之一,hMSCs與DBM共同培養(yǎng),與DBM釋放的可溶性骨生長因子接觸后,能加速自身的增殖與成骨分化。 脈沖電磁場(pulsed electromagnetic fields, PEMF)在矯形外科領(lǐng)域主要用于治療骨不連、骨延遲愈合及假關(guān)節(jié)形成等并已經(jīng)取得成功。特定頻率、特定場強(qiáng)的PEMF能夠刺激成骨細(xì)胞增殖、分化,并可刺激局部骨生長因子的產(chǎn)生,加速骨基質(zhì)礦化,該現(xiàn)象已被國內(nèi)外學(xué)者們證實(shí)。然而有關(guān)PEMF和DBM共同刺激hMSCs增殖與成骨分化的具體生物學(xué)作用研究尚未見報(bào)道。 本研究將觀察PEMF對DBM誘導(dǎo)hMSCs在體外培養(yǎng)時(shí)細(xì)胞增殖與成骨分化的影響,探討PEMF影響組織工程材料中hMSCs成骨分化的可能機(jī)制,為骨組織工程提供新的技術(shù)手段。 方法: 1.經(jīng)知情同意后,選取因股骨頸骨折行髖關(guān)節(jié)置換術(shù)患者的股骨頭。 2.將股骨頭鋸成3mm×5mm×5mm大小的骨粒,按Urist b法制作成DBM。用掃描電鏡觀察材料的形態(tài)并測量材料孔徑,能譜儀檢測DBM鈣含量。 3.獲取健康志愿者的骨髓,分離、培養(yǎng)hMSCs,進(jìn)行原代和傳代培養(yǎng)。鑒定hMSCs的表面抗原標(biāo)志物CD44和CD105。 4.第3代hMSCs以1×104個(gè)/孔接種至8塊24孔板,以1×105個(gè)/孔接種至2塊加蓋玻片的6孔板,各分成4組:細(xì)胞對照組(cell, C組)、細(xì)胞+材料組(cell-DBM, CD組)、細(xì)胞+脈沖電磁場組(cell-PEMF, CP組)、細(xì)胞+材料+脈沖電磁場組(cell-DBM-PEMF, CDP組),其中CD組和CDP組培養(yǎng)板每孔放入一塊DBM材料,CP組和CDP組給予PEMF(4h/d,頻率15Hz,場強(qiáng)5Gs)照射。 5.在第1、3、7、10、14天時(shí)用MTT法分析細(xì)胞的增殖活性;在第1、7、14、21天時(shí)采用ELISA方法檢測堿性磷酸酶(alkaline phosphatase, ALP)活性、放免法檢測骨鈣素(osteocalcin, OC)濃度。 6.在第21天時(shí),進(jìn)行鈣結(jié)節(jié)茜素紅染色,然后在Olympus顯微鏡(IX71型,日本)下進(jìn)行組織學(xué)觀察,并統(tǒng)計(jì)鈣結(jié)節(jié)數(shù)量。 結(jié)果: 1.制作的DBM顏色淡黃,質(zhì)地較軟。掃描電鏡下可見DBM孔隙表面較光滑,孔隙大小為115.4μm-188.4μm,平均孔徑為140.85±21.75μm。能譜儀檢測DBM鈣含量為11.49±0.84%。 2.分離的原代hMSCs呈圓形,24h后可見細(xì)胞貼壁,呈梭形生長;當(dāng)hMSCs生長達(dá)80%融合后,呈同向性生長,旋渦狀排列,細(xì)胞細(xì)長而有突起。hMSCs的表面抗原CD44、CD105免疫組化染色陽性,細(xì)胞膜呈棕褐色。 3.根據(jù)MTT檢測結(jié)果,CD組、CP組和CDP組生長曲線較C組前移,CDP組較C組、CD組、CP組有更高的增殖峰值(p 0.01);在第7、10、14天時(shí),CDP組較C組、CD組、CP組升高(P 0.01),在第7、10天時(shí),CD組較CP組升高(P 0.01)。 4.種植后1天,各組間ALP活性無明顯差異。在種植7天后CD組、CP組、CDP組ALP活性均明顯升高(p 0.01),隨即CP組開始持續(xù)下降,而14天時(shí)CD組和CDP組ALP活性才達(dá)到最高值。第7、14天時(shí)CDP組ALP活性均較CD組、CP組顯著升高(p 0.01),第14天時(shí)CD組ALP活性較CP組顯著升高(p 0.01)。在21天時(shí)CD組、CP組、CDP組組間ALP活性無顯著性差異,但以上三組仍較C組顯著升高(p 0.01)。 5.除C組外,CD組、CP組和CDP組的OC值在種植7天后均持續(xù)顯著增高(p 0.01),其中CDP組增速最快,CD組和CP組OC值在前14天培養(yǎng)過程中均無顯著性差異,第21天時(shí)CD組較CP組顯著升高。CDP組OC值在7、14、21天時(shí)均較CD組、CP組顯著升高(p 0.01)。 6.在21天時(shí)鈣結(jié)節(jié)茜素紅染色在40倍鏡下觀察,平均視野內(nèi)形成的鈣結(jié)節(jié)數(shù)目為C組0個(gè),CD組13.50±1.93個(gè),CP組9.88±1.55個(gè),CDP組19.13±3.27,鈣結(jié)節(jié)數(shù)量CDP組明顯要多于另外三組(p 0.01), CD組多于CP組(p 0.01)。 結(jié)論: 1. DBM與PEMF聯(lián)合刺激及DBM和PEMF單獨(dú)刺激均加快hMSCs的增殖;DBM促hMSCs增殖能力強(qiáng)于PEMF;DBM與PEMF聯(lián)合刺激hMSCs增殖較DBM和PEMF單獨(dú)刺激更強(qiáng)。 2. DBM和PEMF單獨(dú)刺激促進(jìn)hMSCs成骨分化的時(shí)間過程基本一致,但DBM的促分化作用更強(qiáng);DBM與PEMF聯(lián)合對hMSCs的促分化作用較DBM和PEMF單獨(dú)刺激不僅開始時(shí)間早,而且強(qiáng)度更大。 3.在DBM和PEMF單獨(dú)作用促進(jìn)鈣鹽沉積的后期,DBM的作用更強(qiáng);DBM與PEMF聯(lián)合刺激促進(jìn)hMSCs成骨分化鈣鹽沉積時(shí),不僅強(qiáng)度大而且持續(xù)時(shí)間更長。 4. PEMF對DBM誘導(dǎo)hMSCs增殖與成骨分化具有明顯的協(xié)同效應(yīng),外源性的電磁場可以用于骨組織工程促進(jìn)種子細(xì)胞的分化和成骨。
[Abstract]:Background and objective: the decalcified bone matrix (Demineralized Bone Matrix, DBM) with in vitro promotes human bone marrow mesenchymal stem cells (human Mesenchymal Stem Cells, hMSCs) proliferation and osteogenic differentiation in vivo, promote new bone formation, in clinical tissue engineering has wide application value with.HMSCs cell differentiation into bone good and osteogenic activity, is one of the best choice of seed cells for bone tissue engineering, co culture of hMSCs and DBM, growth factor and soluble bone contact DBM release, can accelerate the proliferation and osteogenic differentiation.
Pulsed electromagnetic fields (pulsed electromagnetic fields, PEMF) in the field of orthopaedic surgery is mainly used for the treatment of bone nonunion, bone nonunion and pseudarthrosis and have been successful. Specific frequency, specific field PEMF can stimulate proliferation and differentiation of bone cells, and can stimulate local production of bone growth factors, accelerate the mineralization of bone matrix. This phenomenon has been confirmed by domestic and foreign scholars. However, the specific biological function of research on PEMF and DBM stimulated hMSCs proliferation and osteogenic differentiation has not been reported.
This study will observe the effect of PEMF on the proliferation and osteogenic differentiation of DBM induced hMSCs in vitro, and explore the possible mechanism of PEMF affecting the osteogenic differentiation of hMSCs in tissue-engineered materials, so as to provide new technical means for bone tissue engineering.
Method:
1. after informed consent, the femoral head was selected for the hip arthroplasty for the femoral neck fracture.
2., the femoral head was sawed into the size of 3mm * 5mm * 5mm. The DBM. was made according to Urist B method. The morphology of the material was observed by scanning electron microscope, and the pore diameter of the material was measured. The calcium content of DBM was detected by EDS.
3. obtain the bone marrow of healthy volunteers, separate, culture hMSCs, carry out primary and subculture. Identify the surface antigen markers of hMSCs, CD44 and CD105.
The third generation of hMSCs 4. with a 1 x 104 / 8 holes inoculated to 24 well plates with 1 * 105 / 6 hole plate hole inoculation to 2 pieces with glass, each divided into 4 groups: control group (cell cells, C cells + group), material group (cell-DBM, CD), cell + pulse electromagnetic field group (cell-PEMF, group CP), cells + materials + pulsed electromagnetic field group (cell-DBM-PEMF, group CDP), CD group and CDP group were cultured in each hole into a piece of material DBM, CP group and CDP group were given PEMF (4h/d, frequency 15Hz, field 5Gs) irradiation.
5. on the day of 1,3,7,10,14, the proliferative activity of cells was analyzed by MTT method. The activity of alkaline phosphatase (alkaline phosphatase ALP) was detected by ELISA on day 1,7,14,21, and the concentration of osteocalcin (osteocalcin, OC) was detected by radioimmunoassay.
6. at twenty-first days, calcium nodules were stained with alizarin red, then the histological observation was performed under the Olympus microscope (IX71, Japan), and the number of calcium nodules was counted.
Result:
The color of DBM made of 1. is light yellow and soft. Under scanning electron microscope, the DBM pore surface is smoother, the pore size is 115.4 m-188.4 m-188.4 m, and the average pore size is 140.85 + 21.75 m.. The DBM content of DBM is 11.49 + 0.84%. by EDS.
2., the primary hMSCs of the isolate was round. After 24h, the cells adhered to the wall and grew in spindle shape. When the hMSCs growth reached 80% fusion, the cells grew in the same direction, whirlpool arranged, the cells were elongated and the surface antigen CD44 of.HMSCs was raised, CD105 was positive for immunohistochemical staining, and the cell membrane was brown.
3. according to the results of MTT test, the growth curve of group CD, group CP and group CDP moved forward compared with C group. The CDP group had a higher peak value of proliferation than the C group, CD group and CP group (P 0.01). On the first day of 7,10,14, the group of CP increased (0.01), and the level of group a increased (CP 0.01).
1 4. days after planting, there was no significant difference between the activity of ALP in 7 days after planting. CD group, CP group, CDP group, ALP activity increased significantly (P 0.01), and group CP continued to decline, and 14 days in CD group and CDP group ALP activity was reached the highest value of the first 7,14 days CDP. The activity of ALP were lower than CD group, CP group increased significantly (P 0.01), CD in ALP group was significantly higher than that of CP group at day fourteenth (P 0.01). On the 21 day in CD group, CP group, CDP group has no significant difference between the groups of ALP activity, but more than three were still significantly compared with C group increased (P 0.01).
5. except for C group, CD group, CP group and CDP group OC values at 7 days after planting were significantly higher (P 0.01), the CDP group has the fastest growth rate, CD group and CP group OC in the 14 days before the train had no significant difference during the twenty-first day CD group than in CP the value of OC increased significantly in group.CDP group in 7,14,21 days when compared with CD group, CP group increased significantly (P 0.01).
6., on the 21 day, calcium nodules and alizarin red staining were observed under 40 times microscope. The number of calcium nodules formed in the average field was 0 in group C, 13.50 in group CD, 1.93 in group CD, 9.88 in group CP, 1.55 in group CP, 19.13 19.13 in 3.27 group, and the number of calcium nodules in CDP group was significantly more than that in other three group (P three), and CD group was more than CP group (P P).
Conclusion:
1., the combined stimulation of DBM and PEMF and DBM and PEMF alone stimulated the proliferation of hMSCs, DBM promoted hMSCs proliferation ability stronger than PEMF, and DBM and PEMF stimulated the proliferation of hMSCs more strongly than those of both DBM and H.
2., the time course of promoting hMSCs osteogenesis by DBM and PEMF alone is basically the same, but DBM promotes differentiation. The combination of DBM and PEMF promotes the differentiation of hMSCs more than that of DBM and PEMF alone.
3., in the late stage of DBM and PEMF promoting calcium deposition alone, DBM plays a more powerful role. The joint stimulation of DBM and PEMF promotes hMSCs osteogenesis and calcium deposition.
4. PEMF has obvious synergistic effect on DBM induced hMSCs proliferation and osteogenic differentiation. Exogenous electromagnetic fields can be used in bone tissue engineering to promote differentiation and osteogenesis of seed cells.

【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：R329

【參考文獻(xiàn)】

相關(guān)期刊論文 前8條

1 趙廷寶,范清宇,張殿忠,文艷華;人脫鈣骨基質(zhì)顆粒的制備及應(yīng)注意的問題[J];濱州醫(yī)學(xué)院學(xué)報(bào);2001年01期

2 王忠;高毅;汪艷;潘明新;;人骨髓間充質(zhì)干細(xì)胞的分離培養(yǎng)與鑒定[J];中華神經(jīng)醫(yī)學(xué)雜志;2006年10期

3 沈偉偉;趙建華;;脈沖電磁場對廢用性骨質(zhì)疏松大鼠骨密度和血清轉(zhuǎn)化生長因子β_1的影響[J];第三軍醫(yī)大學(xué)學(xué)報(bào);2009年10期

4 徐巧玲,路麗華,羅二平,申廣浩,劉冬煥,化前珍,吳曉明,李麗榮;低強(qiáng)度脈沖電磁場對大鼠骨密度及骨形態(tài)計(jì)量學(xué)的影響[J];第四軍醫(yī)大學(xué)學(xué)報(bào);2004年21期

5 莫湘濤;骨組織工程支架材料的降解和生物力學(xué)特性[J];醫(yī)用生物力學(xué);2004年01期

6 朱灝,許建中,周強(qiáng),王序全,單建林,趙敏,秦輝;不同年齡人骨髓間充質(zhì)干細(xì)胞體外增殖及成骨分化的研究[J];中國矯形外科雜志;2004年10期

7 王楊;李秀群;黃云;李次會(huì);解慧琪;陳曉禾;鄧力;李馴虎;;不同方法制備生物衍生骨體內(nèi)植入的組織學(xué)觀察[J];中國修復(fù)重建外科雜志;2007年09期

8 梁新剛;楊志明;項(xiàng)舟;李秀群;黃光平;智偉;;異種脫鈣骨基質(zhì)顆粒的生物相容性實(shí)驗(yàn)研究[J];中國修復(fù)重建外科雜志;2009年01期

，

本文編號(hào)：1435792