發(fā)布時間：2018-04-13 23:17

  本文選題：皮質(zhì)骨 + 成骨細(xì)胞��； 參考：《福建中醫(yī)學(xué)院》2009年碩士論文

【摘要】： 目的:通過比較皮質(zhì)骨和其它不同來源兔成骨細(xì)胞的體外培養(yǎng)和部分成骨功能,鑒定并探討不同來源成骨細(xì)胞的成骨能力,通過皮質(zhì)骨塊連續(xù)兩次植塊培養(yǎng)和相關(guān)檢測,探討皮質(zhì)骨連續(xù)植塊培養(yǎng)的可行性。 方法:手術(shù)取3月齡新西蘭大白兔(雌雄不限)上肢皮質(zhì)骨、髂骨、骨膜、骨髓分別用組織塊法和酶消化法培養(yǎng)獲取細(xì)胞,骨髓采用密度梯度離心法提取干細(xì)胞、檢測時經(jīng)成骨誘導(dǎo),每天用倒置相差顯微鏡觀察細(xì)胞形態(tài)、貼壁及生長情況,并用差速貼壁法純化細(xì)胞。將細(xì)胞分為皮質(zhì)骨組、骨膜組、松質(zhì)骨組、干細(xì)胞誘導(dǎo)組和干細(xì)胞常規(guī)組。取不同來源的第3代(P3)細(xì)胞進(jìn)行如下實(shí)驗(yàn),結(jié)晶紫染色比較細(xì)胞形態(tài)、MTT法測定細(xì)胞增殖曲線、對硝基酚法測堿性磷酸酶(ALP)活性、酶免法測定骨鈣素(BGP)等,取原代培養(yǎng)傳代后的皮質(zhì)骨塊進(jìn)行二次組織塊培養(yǎng),獲取第4代(P4)細(xì)胞進(jìn)行實(shí)驗(yàn),比較兩次連續(xù)植塊細(xì)胞的增殖和ALP、BGP表達(dá)能力,多余的細(xì)胞進(jìn)行凍存保種。將各種數(shù)據(jù)進(jìn)行統(tǒng)計學(xué)分析,以均數(shù)±標(biāo)準(zhǔn)差((?)±s)表示,采用成組t檢驗(yàn)和單因素方差分析檢驗(yàn),P＜0.05有統(tǒng)計學(xué)意義。 結(jié)果: 1、形態(tài)學(xué)觀察:不同來源的細(xì)胞形態(tài)差別不大。原代培養(yǎng)松質(zhì)骨組細(xì)胞和皮質(zhì)骨組細(xì)胞基本呈長梭形、放射狀排列于組織塊周圍,骨膜組細(xì)胞呈梭形、三角形,部分松質(zhì)骨組細(xì)胞胞體稍扁、突起較長,皮質(zhì)骨組細(xì)胞突起略短,骨膜組細(xì)胞突起偏短,干細(xì)胞形狀不規(guī)則,呈梭形、多邊形,突起較多、較短。傳代后不同來源的細(xì)胞形態(tài)趨于一致,干細(xì)胞經(jīng)誘導(dǎo)后形狀更加規(guī)則,和其他來源的細(xì)胞類似,呈長梭形、角形及不規(guī)則形等。皮質(zhì)骨兩次植塊培養(yǎng)的細(xì)胞形態(tài)學(xué)上無明顯差異。 2、增殖情況:不同來源的P3細(xì)胞生長呈一致的類S型曲線;細(xì)胞倍增時間,骨膜組和干細(xì)胞常規(guī)組約為5天,干細(xì)胞誘導(dǎo)組為7天,皮質(zhì)骨和松質(zhì)骨組介于其中,為6天,然后逐漸進(jìn)入平臺期。增殖速度,第1天,各組無差別(P＞0.05),第3天開始干細(xì)胞誘導(dǎo)組即落后于皮質(zhì)骨組、骨膜組、松質(zhì)骨組和干細(xì)胞常規(guī)組(P＜0.05),而后四組之間并無明顯差別(P＞0.05),第5天和第7天,皮質(zhì)骨組和松質(zhì)骨組仍無差別(P＞0.05),但二者均落后于骨膜組和干細(xì)胞常規(guī)組,其中只有松質(zhì)骨組落后于骨膜組和干細(xì)胞常規(guī)組有統(tǒng)計學(xué)意義(P＜0.05),骨膜組和干細(xì)胞常規(guī)組無增殖差別(P＞0.05);皮質(zhì)骨兩次連續(xù)植塊細(xì)胞的增殖速度無明顯差異(P＞0.05)。 3、成骨能力: (1)堿性磷酸酶(ALP)活性:皮質(zhì)骨組、骨膜組、松質(zhì)骨組和干細(xì)胞誘導(dǎo)組隨著培養(yǎng)時間的延長,吸光值增大,即ALP活性均增高,培養(yǎng)初中期增殖幅度逐漸增加,后期增幅減慢,而干細(xì)胞常規(guī)組則未見明顯ALP表達(dá)。皮質(zhì)骨組、骨膜組、松質(zhì)骨組和干細(xì)胞誘導(dǎo)組的ALP活性比較:第7天,骨膜組即高于松質(zhì)骨組、干細(xì)胞誘導(dǎo)組(P＜0.05),骨膜組和皮質(zhì)骨組之間、松質(zhì)骨組和干細(xì)胞誘導(dǎo)組之間均無明顯差異(P＞0.05);第12天,骨膜組＞皮質(zhì)骨組＞松質(zhì)骨組＞干細(xì)胞誘導(dǎo)組(P＜0.05);第20天,骨膜組和皮質(zhì)骨組無統(tǒng)計學(xué)差異(P＞0.05),二者均強(qiáng)于松質(zhì)骨組和干細(xì)胞誘導(dǎo)組(P＜0.05),其中松質(zhì)骨組＞干細(xì)胞誘導(dǎo)組(P＜0.05);第28天,骨膜組＞皮質(zhì)骨組＞松質(zhì)骨組＞干細(xì)胞誘導(dǎo)組(P＜0.05)。皮質(zhì)骨兩次連續(xù)植塊細(xì)胞的ALP表達(dá)無明顯差異(P＞0.05)。 (2)骨鈣素(BGP)活性:皮質(zhì)骨組、骨膜組、松質(zhì)骨組和干細(xì)胞誘導(dǎo)組的BGP活性亦隨時間而增強(qiáng),隨培養(yǎng)時間延長而增殖幅度逐漸增加,干細(xì)胞常規(guī)組基本沒有表達(dá)BGP;第12、20天,骨膜組＞皮質(zhì)骨組＞松質(zhì)骨組＞干細(xì)胞誘導(dǎo)組(P＜0.05);第28天,骨膜組＞松質(zhì)骨組＞干細(xì)胞誘導(dǎo)組(P＜0.05),其中,骨膜組和皮質(zhì)骨組之間、皮質(zhì)骨組和松質(zhì)骨組之間均無統(tǒng)計學(xué)差異(P＞0.05)。皮質(zhì)骨兩次連續(xù)植塊細(xì)胞的BGP活性無明顯差異(P＞0.05)。 結(jié)論: 1、皮質(zhì)骨、骨膜、松質(zhì)骨、骨髓干細(xì)胞均可以培養(yǎng)出成骨細(xì)胞。不同來源的成骨細(xì)胞形態(tài)學(xué)上無顯著差別,呈長梭形、三角形或者不規(guī)則形。 2、不同來源的細(xì)胞增殖能力比較如下:骨膜組和干細(xì)胞常規(guī)組增殖較快,皮質(zhì)骨組和松質(zhì)骨組居中(二者之間無統(tǒng)計學(xué)差別),干細(xì)胞誘導(dǎo)組增殖最慢。干細(xì)胞常規(guī)組無明顯成骨能力。不同來源的成骨細(xì)胞成骨能力比較如下:骨膜組＞皮質(zhì)骨組＞松質(zhì)骨組＞干細(xì)胞誘導(dǎo)組。 3、皮質(zhì)骨來源的成骨細(xì)胞成分相對單純,兩次連續(xù)植塊細(xì)胞的生物學(xué)特性無差別,可以進(jìn)行連續(xù)植塊培養(yǎng)以重復(fù)利用骨塊,達(dá)到節(jié)省經(jīng)費(fèi)、節(jié)約時間的目的。
[Abstract]:Objective: To compare the cortical bone and other different sources of rabbit osteoblasts in vitro and osteoblast function, identify and explore different sources of osteogenic ability of osteoblasts, the cortical bone block two continuous explant culture and related testing, to explore the feasibility of cortical bone graft blocks continuous culture.
Methods: the operation of 3 month old New Zealand white rabbits (male or female) upper limb cortical bone, bone, periosteum, bone marrow respectively using tissue explant and enzyme digestion method and cultured bone marrow cells by density gradient centrifugation to extract stem cells, detection after osteogenic induction, every day by the inverted microscope to observe cell morphology, adhesion and the growth, and using differential adhesion purified cells. The cells were divided into cortical bone, periosteum group, cancellous bone group, stem cells group and conventional group. Stem cells from different third generation (P3) cells in the following experiment, comparison of cell morphology and crystal violet staining, cell proliferation assay MTT curve method, p-nitrophenol method of alkaline phosphatase (ALP) activity, enzyme immunoassay method for the determination of osteocalcin (BGP), cultured cortical bone after passage two tissue culture, obtain the fourth generation (P4) cells in experiment, comparison of two continuous explant cells The proliferation and expression of ALP, BGP and redundant cells were cryopreserved. The data were statistically analyzed with mean + standard deviation ((?) s). Group t test and one-way ANOVA test showed that P < 0.05 had statistical significance.
Result:
1 Morphological Observation: cell morphological differences between different sources. Primary culture of cancellous bone and cortical bone cells were fusiform, arranged radially from the tissues, periosteum group cells were spindle shaped, triangular, part of cancellous bone cells slightly flat, long protuberances, cortex bone cells protruding slightly shorter, shorter cell processes in periosteum group, stem cells, irregular shape, fusiform, polygon, projection more shorter. After passage of different sources of cell morphology consistent, stem cells were induced after regular shape, similar to other cells, fusiform, angle shape or irregular shape. No significant difference of cell morphology of cortical bone explant cultured on the two.
2,澧炴畺鎯呭喌:涓嶅悓鏉ユ簮鐨凱3緇嗚優(yōu)鐢熼暱鍛堜竴鑷寸殑綾籗鍨嬫洸綰，

本文編號：1746656