骨髓間充質(zhì)干細(xì)胞和體細(xì)胞的共微囊化研究
發(fā)布時(shí)間：2018-08-26 15:30
【摘要】：骨髓間充質(zhì)干細(xì)胞(BMSCs)來(lái)源豐富、易分離純化且具有多向分化潛能、可自體移植,是作為種子細(xì)胞移植的研究熱點(diǎn)。微膠囊具有良好的免疫隔離性能和生物相容性,為干細(xì)胞大規(guī)模、高活性體外培養(yǎng)及長(zhǎng)期保存提供了技術(shù)支持。共微囊化技術(shù)則利用干細(xì)胞與體細(xì)胞間的相互接觸與支持作用為細(xì)胞移植療法開(kāi)辟了一種新途徑。本文制備了載BMSCs細(xì)胞的海藻酸鈣-幾丁聚糖-海藻酸鈣(ACA)微膠囊,考察了其理化力學(xué)性能及生物相容性。共微囊化BMSCs/肝細(xì)胞(Chang liver),考察細(xì)胞代謝性能。共微囊化BMSCs/胰島β細(xì)胞(Beta-TC-6)用于治療Ⅰ型糖尿病,并在分子水平探討其機(jī)理。 首先,體外培養(yǎng)BMSCs細(xì)胞,繪制其細(xì)胞生長(zhǎng)曲線。然后采用免疫細(xì)胞組織化學(xué)染色方法鑒定其表面抗原CD29、CD44、CD117,檢測(cè)結(jié)果為CD29呈陽(yáng)性,CD44呈陽(yáng)性,CD117呈陰性,成骨誘導(dǎo)分化實(shí)驗(yàn)結(jié)果也表明BMSCs在體外培養(yǎng)時(shí)具有分化的能力。 其次,采用高壓靜電法制備載BMSCs細(xì)胞的ACA微膠囊,優(yōu)化制備工藝為:兩步法制備,海藻酸鈉濃度1.75%(w/v),成膜時(shí)間10~ min,細(xì)胞密度1×10~6個(gè)/mL。制出的載BMSCs微囊球形度好、表面光滑,粒徑主要分布在300~400μm間且7天內(nèi)隨時(shí)間變化不大,機(jī)械強(qiáng)度較好且囊內(nèi)細(xì)胞分布均勻、活性高,破囊后重新培養(yǎng)的BMSCs細(xì)胞生長(zhǎng)良好。 再次,考察體外培養(yǎng)的微囊化BMSCs細(xì)胞的活性(CCK-8法),發(fā)現(xiàn)與平板培養(yǎng)和非液化核心的微囊化細(xì)胞相比,液化核心的微囊更適于細(xì)胞的長(zhǎng)期生長(zhǎng)。體外培養(yǎng)的第15天,細(xì)胞密度達(dá)到峰值為4.3×10~7個(gè)/mL。將載BMSCs細(xì)胞的微囊植入小鼠腹腔考察微膠囊的體內(nèi)生物相容性,觀察發(fā)現(xiàn)小鼠生理活動(dòng)正常,未見(jiàn)毒性癥狀,體重增長(zhǎng)平穩(wěn)。28天內(nèi)回收微囊,微囊表面光滑,球形度好,未出現(xiàn)纖維化或巨噬細(xì)胞聚集現(xiàn)象。 然后,采用平板、微囊化、共培養(yǎng)及共微囊化四種培養(yǎng)方式來(lái)培養(yǎng)肝細(xì)胞(Chang liver),以白蛋白及尿素分泌量為檢測(cè)指標(biāo)考察比較肝細(xì)胞的代謝性能。結(jié)果表明,四組分泌白蛋白量及尿素的大小關(guān)系均為:共微囊化BMSCs/Chang liver組 BMSCs/Chang liver組微囊化Chang liver組 Chang liver細(xì)胞組。15天內(nèi),共微囊化BMSCs/Chang liver組白蛋白分泌量最高為3.08 mg/mL。11天內(nèi),尿素分泌量最高為3.75 mmol/L。 最后,制備微囊化胰島β細(xì)胞(Beta-TC-6)及共微囊化BMSCs/Beta-TC-6細(xì)胞,測(cè)定比較28天內(nèi)胰島素分泌量。第28天,共微囊化組分泌胰島素含量為98.60 pg/mL。將兩種微囊化細(xì)胞植入Ⅰ型糖尿病小鼠體內(nèi),監(jiān)測(cè)比較其降血糖作用。結(jié)果表明,兩者均能在28天內(nèi)明顯降低糖尿病小鼠的血糖,且共微囊化組具有更良好的效果,移植后的第28天,微囊化Beta-TC-6細(xì)胞組及共微囊化BMSCs/Beta-TC-6細(xì)胞組糖尿病小鼠血糖濃度分別為5.08 mmol/L和6.08 mmol/L。采用RT-PCR的方法檢測(cè)細(xì)胞胰島素mRNA表達(dá)水平,結(jié)果表明BMSCs與胰島β細(xì)胞在微囊微環(huán)境中共培養(yǎng)后能在其誘導(dǎo)下在一定程度上分化為胰島素分泌細(xì)胞。 本研究證明了微膠囊可以為骨髓間充質(zhì)干細(xì)胞誘導(dǎo)分化成成體細(xì)胞提供微環(huán)境,共微囊化骨髓間充質(zhì)干細(xì)胞和體細(xì)胞用于移植治療疾病具有潛在應(yīng)用前景。
[Abstract]:Bone marrow mesenchymal stem cells (BMSCs) are abundant in origin, easy to isolate and purify, and have multi-directional differentiation potential. They can be transplanted by themselves. Microencapsulation has good immunoisolation and biocompatibility, which provides technical support for large-scale, high-activity in vitro culture and long-term preservation of stem cells. In this paper, calcium alginate-chitosan-calcium alginate (ACA) microcapsules loaded with BMSCs cells were prepared and their physical and chemical properties and biocompatibility were investigated. Metabolic properties of cells. Co-microencapsulated BMSCs/islet beta cells (Beta-TC-6) were used to treat type 1 diabetes mellitus and the mechanism was discussed at the molecular level.
Firstly, BMSCs cells were cultured in vitro and their growth curves were drawn. Then the surface antigens CD29, CD44 and CD117 were identified by immunocytochemical staining. The results showed that CD29, CD44 and CD117 were positive, CD44 were positive and CD117 were negative.
Secondly, the ACA microcapsules loaded with BMSCs cells were prepared by high-voltage electrostatic method. The optimized preparation process was as follows: sodium alginate concentration 1.75% (w/v), film forming time 10-min, cell density 1 x106 / mL. The prepared BMSCs-loaded microcapsules had good sphericity, smooth surface, mainly distributed between 300-400 microns and had little change with time within 7 days, and mechanical properties. BMSCs cells grew well after burbreaking.
Thirdly, the activity of microencapsulated BMSCs cells cultured in vitro (CCK-8 method) was investigated, and it was found that the microcapsules with liquefied core were more suitable for the long-term growth of cells than those with non-liquefied core and plate culture. The biocompatibility of the microcapsules in vivo was observed. The mice showed normal physiological activities, no toxic symptoms and stable weight gain. Within 28 days, the microcapsules were recovered. The surface of the microcapsules was smooth and spherical, and no fibrosis or macrophage aggregation was observed.
Then, Chang liver cells were cultured by plate, microencapsulation, co-culture and co-microencapsulation. The metabolic properties of hepatocytes were evaluated by the levels of albumin and urea. The results showed that the relationship between the amount of albumin and urea in the four groups was as follows: BMSCs/Changlive group BMSCs/Chang. The highest albumin secretion and urea secretion were 3.08 mg/mL and 3.75 mmol/L in the co-microencapsulated BMSCs/Changlive group within 15 days and 3.08 mg/mL respectively.
At last, we prepared microencapsulated islet beta cells (Beta-TC-6) and co-microencapsulated BMSCs/Beta-TC-6 cells, and measured and compared their insulin secretion in 28 days. On the 28th day, the content of insulin secreted by co-microencapsulated fraction was 98.60 pg/mL. Two kinds of microencapsulated cells were implanted into type I diabetic mice to monitor and compare their hypoglycemic effects. The blood glucose levels of diabetic mice in the microencapsulated Beta-TC-6 cell group and the co-microencapsulated BMSCs/Beta-TC-6 cell group were 5.08 mmol/L and 6.08 mmol/L respectively on the 28th day after transplantation. The results showed that BMSCs and islet beta cells could differentiate into insulin-secreting cells to some extent after co-culture in microcapsule microenvironment.
This study demonstrates that microcapsules can provide a microenvironment for the induction and differentiation of bone marrow mesenchymal stem cells into adult cells, and co-microencapsulated bone marrow mesenchymal stem cells and somatic cells have potential applications in transplantation therapy.
【學(xué)位授予單位】：華僑大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類號(hào)】：R329
本文編號(hào)：2205323
[Abstract]:Bone marrow mesenchymal stem cells (BMSCs) are abundant in origin, easy to isolate and purify, and have multi-directional differentiation potential. They can be transplanted by themselves. Microencapsulation has good immunoisolation and biocompatibility, which provides technical support for large-scale, high-activity in vitro culture and long-term preservation of stem cells. In this paper, calcium alginate-chitosan-calcium alginate (ACA) microcapsules loaded with BMSCs cells were prepared and their physical and chemical properties and biocompatibility were investigated. Metabolic properties of cells. Co-microencapsulated BMSCs/islet beta cells (Beta-TC-6) were used to treat type 1 diabetes mellitus and the mechanism was discussed at the molecular level.
Firstly, BMSCs cells were cultured in vitro and their growth curves were drawn. Then the surface antigens CD29, CD44 and CD117 were identified by immunocytochemical staining. The results showed that CD29, CD44 and CD117 were positive, CD44 were positive and CD117 were negative.
Secondly, the ACA microcapsules loaded with BMSCs cells were prepared by high-voltage electrostatic method. The optimized preparation process was as follows: sodium alginate concentration 1.75% (w/v), film forming time 10-min, cell density 1 x106 / mL. The prepared BMSCs-loaded microcapsules had good sphericity, smooth surface, mainly distributed between 300-400 microns and had little change with time within 7 days, and mechanical properties. BMSCs cells grew well after burbreaking.
Thirdly, the activity of microencapsulated BMSCs cells cultured in vitro (CCK-8 method) was investigated, and it was found that the microcapsules with liquefied core were more suitable for the long-term growth of cells than those with non-liquefied core and plate culture. The biocompatibility of the microcapsules in vivo was observed. The mice showed normal physiological activities, no toxic symptoms and stable weight gain. Within 28 days, the microcapsules were recovered. The surface of the microcapsules was smooth and spherical, and no fibrosis or macrophage aggregation was observed.
Then, Chang liver cells were cultured by plate, microencapsulation, co-culture and co-microencapsulation. The metabolic properties of hepatocytes were evaluated by the levels of albumin and urea. The results showed that the relationship between the amount of albumin and urea in the four groups was as follows: BMSCs/Changlive group BMSCs/Chang. The highest albumin secretion and urea secretion were 3.08 mg/mL and 3.75 mmol/L in the co-microencapsulated BMSCs/Changlive group within 15 days and 3.08 mg/mL respectively.
At last, we prepared microencapsulated islet beta cells (Beta-TC-6) and co-microencapsulated BMSCs/Beta-TC-6 cells, and measured and compared their insulin secretion in 28 days. On the 28th day, the content of insulin secreted by co-microencapsulated fraction was 98.60 pg/mL. Two kinds of microencapsulated cells were implanted into type I diabetic mice to monitor and compare their hypoglycemic effects. The blood glucose levels of diabetic mice in the microencapsulated Beta-TC-6 cell group and the co-microencapsulated BMSCs/Beta-TC-6 cell group were 5.08 mmol/L and 6.08 mmol/L respectively on the 28th day after transplantation. The results showed that BMSCs and islet beta cells could differentiate into insulin-secreting cells to some extent after co-culture in microcapsule microenvironment.
This study demonstrates that microcapsules can provide a microenvironment for the induction and differentiation of bone marrow mesenchymal stem cells into adult cells, and co-microencapsulated bone marrow mesenchymal stem cells and somatic cells have potential applications in transplantation therapy.
【學(xué)位授予單位】：華僑大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類號(hào)】：R329
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