大鼠骨髓间充质干细胞体外培养诱导分化甲状旁腺细胞的研究
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摘要
目的：
研究骨髓间充质干细胞（BMSCs）体外定向分化甲状旁腺细胞的方法。研究表明，骨髓间充质干细胞是一种易于分离、培养、扩增，并具有多项分化潜能的细胞，可分化为骨细胞、脂肪细胞、心肌细胞及干细胞。可充当多种器官重建和损伤修复反应的细胞源。目前，关于间充质干细胞体外培养分化甲状旁腺细胞的研究报道极少。甲状旁腺是人体重要的内分泌腺，甲状旁腺细胞的主要功能是分泌甲状旁腺激素，主要功能是调节并影响人体内钙与磷的代谢。其分泌不足即可导致甲状旁腺功能减退。甲状旁腺功能减退症是甲状腺手术后最常见的并发症，可以引起喉及膈肌痉挛，甚至窒息死亡，严重威胁着人类的健康和生命，目前尚无有效的治疗方法，一直是医学上的一个难点。研究骨髓间充质干细胞定向为分化甲状旁腺细胞，对以后临床治疗甲状旁腺损伤修复及甲状旁腺功能减退症具有重要意义。因此，我们进行间充质干细胞的体外培养，诱导其定向分化为甲状旁腺细胞，研究分化方法，为进一步的临床治疗甲状旁腺功能减退症奠定一定基础。
方法：
1周龄SD大鼠脱颈处死后，置于75%乙醇中浸泡15min，在无菌条件下取出大鼠股骨、胫骨，分离、纯化BMSCs，在体外进行培养、传代。应用流式细胞仪检测细胞表面标记（CD44、CD45、CD90），对BMSCs进行鉴定。诱导BMSCs向神经细胞分化成功，表明本方法获得的BMSCs能跨胚层分化，具有多向分化潜能。切取大鼠甲状旁腺，置入4℃RPMI1640培养液中（含15%胎牛血清），剪碎、漂洗、离心法培养甲状旁腺细，作为阳性对照组。再制备甲状旁腺无细胞裂解液。然后将其分为2个对照组，1组加入含100ng·mL-1ActivinA、100ng·mL-1SHH、100μL·mL-1甲状旁腺无细胞裂解液和0.2%FBS的DMEM-F12培养液继续培养，2组则不加入甲状旁腺无细胞裂解液，1d后增加血清浓度为2%培养，1d后后增加血清浓度为5%继续培养。诱导骨髓间充干细胞分化甲状旁腺细胞。分别于5d、12d、19d和26d后收集细胞，通过免疫细胞化学、免疫荧光化学、Western blot、PCR方法检测甲状旁腺细胞表面标志物（GCM2、CASR、PTH）的表达，对分化的甲状旁腺细胞进行鉴定。
结果：
①显微镜下可观察到传代后均一性较好的BMSCs；体外培养的BMSCs经诱导，分化成为神经元样细胞，GFAP和MAP2免疫细胞化学和免疫荧光染色呈阳性表达；②加入甲状旁腺无细胞裂解液的对照组均能收获得较好的甲状旁腺细胞分化效果，分化后的甲状旁腺细胞的标志物CASR、GCM2、PTH的表达与甲状旁腺细胞阳性对照组进行统计学分析（p＜0.05）。未加入甲状旁腺无细胞裂解液的对照组则分化效果不明显。
结论：
本实验成功提取大鼠骨髓间充质干细胞，经体外培养后，加入Activin A、SHH、甲状旁腺无细胞裂解液和0.2%FBS的DMEM-F12定向诱导可较好的分化甲状旁腺细胞，为今后临床上修复甲状旁腺损伤，及治疗甲状旁腺功能减退症奠定了一定的实验基础。
Objective:
To study the directional differentiation of bone marrowmesenchymal stem cells (BMSCs) into parathyroid cells in vitro. Studieshave shown that bone marrow mesenchymal stem cells is an kind of cellswith easy separation, culture, expansion, and possessing multipledifferentiation potential, which can differentiate into bone cells, fat cells,cardiac cells and stem cells. It can act as the cell source of multiple organreconstruction and damage repair. Currently, there are only few literaturesabout differentiation of mesenchymal stem cells into parathyroid cells invitro. Is an important human parathyroid glands, parathyroid cells is themain function of parathyroid hormone secretion, the main function is toregulate the body and affects the metabolism of calcium and phosphoruspeople. Which can lead to inadequate secretion of hypoparathyroidism.Hypoparathyroidism, which is the most common complications afterthyroid surgery, can cause throat and diaphragm spasm, and evensuffocation death, seriously threatening the health and life human being.There is no effective treatment today, and it has been a difficulty medically. The study on the directional differentiation of bone marrowmesenchymal stem cells into parathyroid cells is of great significance inthe clinical treatment of parathyroid damage and hyperparathyroidism.Therefore, we cultured mesenchymal stem cells in vitro, induced it todifferentiate into parathyroid cells directionally, and studied thedifferentiation method, which laid a certain foundation for further clinicaltreatment of parathyroid damage.
Method:
1-week-old SD rats were sacrificed by cervical vertebra luxation,and placed in75%ethanol for15min. The rat femur and tibia were takenout under sterile conditions. en BMSCs was separated and purified, andcultured for passage in vitro. BMSCs were identified using flowcytometry to detect cell surface markers (CD44, CD45, CD90). BMSCswere induced to differentiate to neural cells, indicating that BMSCsobtained with this method can differentiate into cells of trans-germinallayer, with multi-directional differentiation potentials. Cut rat parathyroid,placed it in RPMI1640culture medium (containing15%fetal bovineserum) at4℃, cut into pieces, rinsed, and cultured the parathyroid withcentrifugation method as a positive control group. Then the parathyroidcell lysate was prepared. Then it was divided into two control groups. Thefirst group was added with parathyroid cell-free lysates containing100ng mL-1Activin A,100ng mL-1SHH,100μL mL-1and DMEM-F12 culture medium containing0.2%FBS and cultured. The second groupwas not added with parathyroid cell free lysates, increased the serumconcentration to2%after1day culture, further increased the serumconcentration to5%after another1day and continued to culture. Bonemarrow mesenchymal stem cells were induced to differentiate toparathyroid cells. Cells were collected at5d,12d,19d and26d,respectively. The differentiated parathyroid cells were identified bydetecting parathyroid cell surface markers (GCM2, CASR, PTH) usingmethods of immunocytochemistry, immunofluorescence, Western blot,and PCR.
Results:
①BMSCs with better uniformity after passage can be observedunder the microscope; BMSCs cultured in vitro were induced todifferentiate into neuron-like cells, in which GFAP, MAP2showedpositive expression with immunocytochemistry and immunofluorescencestaining;②The control group with the parathyroid cell-free lysate couldgain parathyroid cells with better differentiation; the expression ofmarkers CASR, GCM2, PTH in differentiated parathyroid cells and thatof the positive control group were made a statistical analysis (p <0.05).The differentiation effect of control group without parathyroid cell-freelysate was not obvious.
Conclusion:
The rat bone marrow mesenchymal stem cells were extractedsuccessfully, after culturing in vitro, Activin A, SHH, parathyroidcell-free lysates, and0.2%FBS DMEM-F12were added to inducedifferentiation of parathyroid cells to gain a better effect, laying a certainexperimental foundation to repair parathyroid damage clinically and treathypoparathyroidism.
研究骨髓间充质干细胞（BMSCs）体外定向分化甲状旁腺细胞的方法。研究表明，骨髓间充质干细胞是一种易于分离、培养、扩增，并具有多项分化潜能的细胞，可分化为骨细胞、脂肪细胞、心肌细胞及干细胞。可充当多种器官重建和损伤修复反应的细胞源。目前，关于间充质干细胞体外培养分化甲状旁腺细胞的研究报道极少。甲状旁腺是人体重要的内分泌腺，甲状旁腺细胞的主要功能是分泌甲状旁腺激素，主要功能是调节并影响人体内钙与磷的代谢。其分泌不足即可导致甲状旁腺功能减退。甲状旁腺功能减退症是甲状腺手术后最常见的并发症，可以引起喉及膈肌痉挛，甚至窒息死亡，严重威胁着人类的健康和生命，目前尚无有效的治疗方法，一直是医学上的一个难点。研究骨髓间充质干细胞定向为分化甲状旁腺细胞，对以后临床治疗甲状旁腺损伤修复及甲状旁腺功能减退症具有重要意义。因此，我们进行间充质干细胞的体外培养，诱导其定向分化为甲状旁腺细胞，研究分化方法，为进一步的临床治疗甲状旁腺功能减退症奠定一定基础。
方法：
1周龄SD大鼠脱颈处死后，置于75%乙醇中浸泡15min，在无菌条件下取出大鼠股骨、胫骨，分离、纯化BMSCs，在体外进行培养、传代。应用流式细胞仪检测细胞表面标记（CD44、CD45、CD90），对BMSCs进行鉴定。诱导BMSCs向神经细胞分化成功，表明本方法获得的BMSCs能跨胚层分化，具有多向分化潜能。切取大鼠甲状旁腺，置入4℃RPMI1640培养液中（含15%胎牛血清），剪碎、漂洗、离心法培养甲状旁腺细，作为阳性对照组。再制备甲状旁腺无细胞裂解液。然后将其分为2个对照组，1组加入含100ng·mL-1ActivinA、100ng·mL-1SHH、100μL·mL-1甲状旁腺无细胞裂解液和0.2%FBS的DMEM-F12培养液继续培养，2组则不加入甲状旁腺无细胞裂解液，1d后增加血清浓度为2%培养，1d后后增加血清浓度为5%继续培养。诱导骨髓间充干细胞分化甲状旁腺细胞。分别于5d、12d、19d和26d后收集细胞，通过免疫细胞化学、免疫荧光化学、Western blot、PCR方法检测甲状旁腺细胞表面标志物（GCM2、CASR、PTH）的表达，对分化的甲状旁腺细胞进行鉴定。
结果：
①显微镜下可观察到传代后均一性较好的BMSCs；体外培养的BMSCs经诱导，分化成为神经元样细胞，GFAP和MAP2免疫细胞化学和免疫荧光染色呈阳性表达；②加入甲状旁腺无细胞裂解液的对照组均能收获得较好的甲状旁腺细胞分化效果，分化后的甲状旁腺细胞的标志物CASR、GCM2、PTH的表达与甲状旁腺细胞阳性对照组进行统计学分析（p＜0.05）。未加入甲状旁腺无细胞裂解液的对照组则分化效果不明显。
结论：
本实验成功提取大鼠骨髓间充质干细胞，经体外培养后，加入Activin A、SHH、甲状旁腺无细胞裂解液和0.2%FBS的DMEM-F12定向诱导可较好的分化甲状旁腺细胞，为今后临床上修复甲状旁腺损伤，及治疗甲状旁腺功能减退症奠定了一定的实验基础。
Objective:
To study the directional differentiation of bone marrowmesenchymal stem cells (BMSCs) into parathyroid cells in vitro. Studieshave shown that bone marrow mesenchymal stem cells is an kind of cellswith easy separation, culture, expansion, and possessing multipledifferentiation potential, which can differentiate into bone cells, fat cells,cardiac cells and stem cells. It can act as the cell source of multiple organreconstruction and damage repair. Currently, there are only few literaturesabout differentiation of mesenchymal stem cells into parathyroid cells invitro. Is an important human parathyroid glands, parathyroid cells is themain function of parathyroid hormone secretion, the main function is toregulate the body and affects the metabolism of calcium and phosphoruspeople. Which can lead to inadequate secretion of hypoparathyroidism.Hypoparathyroidism, which is the most common complications afterthyroid surgery, can cause throat and diaphragm spasm, and evensuffocation death, seriously threatening the health and life human being.There is no effective treatment today, and it has been a difficulty medically. The study on the directional differentiation of bone marrowmesenchymal stem cells into parathyroid cells is of great significance inthe clinical treatment of parathyroid damage and hyperparathyroidism.Therefore, we cultured mesenchymal stem cells in vitro, induced it todifferentiate into parathyroid cells directionally, and studied thedifferentiation method, which laid a certain foundation for further clinicaltreatment of parathyroid damage.
Method:
1-week-old SD rats were sacrificed by cervical vertebra luxation,and placed in75%ethanol for15min. The rat femur and tibia were takenout under sterile conditions. en BMSCs was separated and purified, andcultured for passage in vitro. BMSCs were identified using flowcytometry to detect cell surface markers (CD44, CD45, CD90). BMSCswere induced to differentiate to neural cells, indicating that BMSCsobtained with this method can differentiate into cells of trans-germinallayer, with multi-directional differentiation potentials. Cut rat parathyroid,placed it in RPMI1640culture medium (containing15%fetal bovineserum) at4℃, cut into pieces, rinsed, and cultured the parathyroid withcentrifugation method as a positive control group. Then the parathyroidcell lysate was prepared. Then it was divided into two control groups. Thefirst group was added with parathyroid cell-free lysates containing100ng mL-1Activin A,100ng mL-1SHH,100μL mL-1and DMEM-F12 culture medium containing0.2%FBS and cultured. The second groupwas not added with parathyroid cell free lysates, increased the serumconcentration to2%after1day culture, further increased the serumconcentration to5%after another1day and continued to culture. Bonemarrow mesenchymal stem cells were induced to differentiate toparathyroid cells. Cells were collected at5d,12d,19d and26d,respectively. The differentiated parathyroid cells were identified bydetecting parathyroid cell surface markers (GCM2, CASR, PTH) usingmethods of immunocytochemistry, immunofluorescence, Western blot,and PCR.
Results:
①BMSCs with better uniformity after passage can be observedunder the microscope; BMSCs cultured in vitro were induced todifferentiate into neuron-like cells, in which GFAP, MAP2showedpositive expression with immunocytochemistry and immunofluorescencestaining;②The control group with the parathyroid cell-free lysate couldgain parathyroid cells with better differentiation; the expression ofmarkers CASR, GCM2, PTH in differentiated parathyroid cells and thatof the positive control group were made a statistical analysis (p <0.05).The differentiation effect of control group without parathyroid cell-freelysate was not obvious.
Conclusion:
The rat bone marrow mesenchymal stem cells were extractedsuccessfully, after culturing in vitro, Activin A, SHH, parathyroidcell-free lysates, and0.2%FBS DMEM-F12were added to inducedifferentiation of parathyroid cells to gain a better effect, laying a certainexperimental foundation to repair parathyroid damage clinically and treathypoparathyroidism.
引文
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