The effect of platelet-rich plasma gels on adipose mesenchymal stem cells overexpressing glia-derived neurotrophic factor
-
摘要:
目的 探讨富血小板血浆（PRP）凝胶对过表达胶质细胞源性神经营养因子（GDNF）的脂肪间充质干细胞（ADSC）的作用。 方法 该研究为实验研究。取5只成年雄性SD大鼠,采用胶原酶消化法获取原代ADSC并进行鉴定。取第3代单纯ADSC,按照随机数字表法（分组方法下同）分为感染空载腺病毒的阴性对照组和感染过表达GDNF腺病毒的过表达GDNF组。培养48 h后,大体观察细胞感染情况。另取5只成年雄性SD大鼠,采血后采用差速离心法获取PRP,制备成凝胶后采用扫描电子显微镜观察其微观结构。取第3代单纯ADSC,加入PRP成胶前的混合液中,成胶后常规培养48 h,行苏木精-伊红染色观察细胞生长情况,行钙黄素/碘化丙啶染色检测细胞活/死情况。取24孔板/共聚焦皿,分为阴性对照凝胶组和过表达GDNF凝胶组,分别加入感染空载腺病毒的ADSC和感染过表达GDNF的ADSC,并在PRP凝胶中进行常规培养。培养48 h后,采用钙黄素/碘化丙啶染色检测细胞活/死情况；培养24、48、72 h及1、2、3、4周后,收集细胞培养基的上清液,采用酶标仪测定其吸光度值并计算GDNF含量,样本数为3；培养48 h后,采用免疫荧光法检测细胞表达S100蛋白质（施万细胞特异性标志物）的情况。 结果 培养48 h后,阴性对照组和过表达GDNF组中感染腺病毒的细胞占比均接近90%,且生长状态良好。阴性对照组细胞正常生长；过表达GDNF组细胞的形态发生明显变化,约80%~90%的细胞伸出2个或多个突起,且在细胞聚集的地方,突起交织成网状。PRP凝胶呈三维网状结构,且孔径大小不一。培养48 h后,单纯ADSC可以很好地附着于PRP凝胶,且活细胞占比达98%。培养48 h,阴性对照凝胶组细胞生长良好,且呈典型的单纯ADSC样梭形生长；过表达GDNF凝胶组细胞生长良好,且多数细胞伸出2个或多个突起,突起聚集处交织成网状。培养24、48、72 h及1、2、3、4周后,过表达GDNF凝胶组细胞培养基的上清液中GDNF含量分别为（90±10）、（133±15）、（150±10）、（137±15）、（132±18）、（120±10）、（127±16）pg/mL,均明显高于阴性对照凝胶组的（42±7）、（44±7）、（43±6）、（47±6）、（49±5）、（49±6）、（51±4）pg/mL,t值分别为6.20、8.08、15.18、9.12、7.99、9.61、7.86,P<0.05。培养48 h后,与阴性对照凝胶组相比,过表达GDNF凝胶组细胞表达S100蛋白质的荧光强度明显增强。 结论 制备的三维PRP凝胶具有良好的生物相容性,可负载过表达GDNF的ADSC并缓释GDNF,诱导ADSC向高表达S100蛋白质的施万细胞分化。 Abstract:Objective To investigate the effect of platelet-rich plasma (PRP) gel on adipose mesenchymal stem cells (ADSCs) overexpressing glial cell-derived neurotrophic factor (GDNF), namely GDNF-ADSCs. Methods This study was an experimental study. Five adult male Sprague-Dawley rats (the same below) were used, and the primary ADSCs were obtained by collagenase digestion, and then the cells were indentified. The 3rd generation ADSCs were obtained and divided into negative control group infected with empty adenovirus and overexpressing GDNF group infected with overexpressing GDNF adenovirus, according to random number table method. After 48 hours of culture, the infection of cells was observed. Five adult male Sprague-Dawley rats (the same below) were used, and the PRP was obtained after collecting blood by differential centrifugation. The microstructure of PRP was observed by scanning electron microscope. The 3rd generation ADSCs were added into the mixture before PRP being gel and cultured for 48 h ours. The cell growth was observed by hematoxylin-eosin staining, and the cell viability/death was detected by calcein/propyl iodide staining. PRP gels loaded with overexpression of GDNF-ADSCs and PRP gels loaded with ADSCs infected with empty adenovirus were prepared. A 24-well plate/confocal dish was divided into negative control gel group and GDNF overexpressed gel group. After 48 hours of culture, the cell viability/death was detected by calcein/propyl iodide staining. After culture for 24, 48, 72 hours and 1, 2, 3, 4 weeks, the supernatant of cell culture medium was collected, the absorbance value was determined by enzyme-labeled analyzer and the GDNF content was calculated, with the sample number of 3. After 48 hours of culture, the expression of S100 protein (a specific marker of Schwann cells) was detected by immunofluorescence assay. Results After 48 hours of culture, the proportions of cells infected with adenovirus in negative control group and overexpressed GDNF group were close to 90%, and the cell growth was good. The cells in negative control group grew normally. The morphology of the cells in overexpressed GDNF group was significantly changed, about 80%-90% of the cells had two or more protruding processes, and the protruding processes were interwoven to form a network where the cells gathered. PRP gel formed a three-dimensional network structure with different pore sizes. After 48 hours of culture, ADSCs could be well attached to PRP gel, and 98% of the cells were alive. After 48 hours of culture, the cells in negative control gel group grew well and showed typical ADSC-like spindle-shaped growth. The cells in overexpressed GDNF gel group grew well, and most of the cells had two or more protrusions, and the protrusions were interwoven into a network. After culture for 24, 48, 72 hours and 1, 2, 3, 4 weeks, the content of GDNF in the supernatant of cell culture medium in overexpressed GDNF gel group was (90±10), (133±15), (150±10), (137±15), (132±18), (120±10), and (127±16) pg/mL, which was significantly higher than (41.67±7.29), (44.00±6.95), (43.00±6.09), (47.00±5.51), (49.00±5.05), (48.67±5.93), and (51.33±4.16) pg/mL in negative control gel group (with t values of 6.20, 8.08, 15.18, 9.12, 7.99, 9.61, and 7.86, respectively, P<0.05). After 48 hours of culture, the fluorescence intensity of S100 protein expression of cells in GDNF overexpressed gel group was significantly stronger than that in negative control gel group. Conclusions The prepared three-dimensional PRP gel has good biocompatibility and can lood GDNF-ADSCs and release GDNF slowly, inducing ADSCs to differentiate into Schwann cells with high S100 protein expression. -
参考文献
(38) [1] SullivanR, DaileyT, DuncanK, et al. Peripheral nerve injury: stem cell therapy and peripheral nerve transfer[J]. Int J Mol Sci, 2016,17(12) :2101. DOI: 10.3390/ijms17122101. [2] NegroS, PirazziniM, RigoniM. Models and methods to study Schwann cells[J]. J Anat, 2022,241(5):1235-1258. DOI: 10.1111/joa.13606. [3] KhanA, DiazA, BrooksAE, et al. Scalable culture techniques to generate large numbers of purified human Schwann cells for clinical trials in human spinal cord and peripheral nerve injuries[J]. J Neurosurg Spine, 2022,36(1):135-144. DOI: 10.3171/2020.11.SPINE201433. [4] ReschA, WolfS, MannA, et al. Co-culturing human adipose derived stem cells and schwann cells on spider silk-a new approach as prerequisite for enhanced nerve regeneration[J]. Int J Mol Sci, 2018,20(1):71. DOI: 10.3390/ijms20010071. [5] 郭洪刚, 姚芳莲, 汪涛, 等. 富血小板血浆联合人重组骨形态形成蛋白-2诱导羊脂肪基质干细胞成骨化趋势的研究[J].中华显微外科杂志,2016,39(2):138-142. DOI: 10.3760/cma.j.issn.1001-2036.2016.02.009. [6] 李绍磊, 杨有优, 刘云江, 等. 携带增强型绿色荧光蛋白的慢病毒载体转染大鼠脂肪干细胞[J].中华显微外科杂志,2014,37(2):147-151. DOI: 10.3760/cma.j.issn.1001-2036.2014.02.013. [7] 张栋鑫, 肖丽玲. 脂肪干细胞联合水凝胶材料在组织工程中的研究进展及前景[J].国际生物医学工程杂志,2021,44(4):323-328. DOI: 10.3760/cma.j.cn121382-20210301-00413. [8] 吴飞, 邓明, 杨越, 等. 复合脂肪源性干细胞的VPA/PRGD组织工程神经修复大鼠坐骨神经缺损的实验研究[J].中华显微外科杂志,2017,40(4):353-357. DOI: 10.3760/cma.j.issn.1001-2036.2017.04.011. [9] 皮刚, 杨大平, 田晓东. ADSCs复合纤维蛋白凝胶修复大鼠坐骨神经缺损[J].中国美容整形外科杂志,2013,24(6):375-378. DOI: 10.3969/j.jssn.1673-7040.2013.06.018. [10] ZhengZ, LiuJ. GDNF-ADSCs-APG embedding enhances sciatic nerve regeneration after electrical injury in a rat model[J]. J Cell Biochem, 2019,120(9):14971-14985. DOI: 10.1002/jcb.28759. [11] 刘海琴, 马华根, 唐元瑜. 原代大鼠脂肪间充质干细胞的体外培养扩增及鉴定[J].中国组织工程研究,2022,26(19):2953-2957. [12] 田新立, 江波, 颜洪. 富血小板血浆对大鼠背部超长随意皮瓣成活的影响[J].中华烧伤杂志,2019,35(1):48-53. DOI: 10.3760/cma.j.issn.1009-2587.2019.01.009. [13] HercherD, NguyenMQ, DworakH. Extracellular vesicles and their role in peripheral nerve regeneration[J]. Exp Neurol, 2022,350:113968. DOI: 10.1016/j.expneurol.2021.113968. [14] ZhangD, NiN, WangY, et al. CircRNA-vgll3 promotes osteogenic differentiation of adipose-derived mesenchymal stem cells via modulating miRNA-dependent integrin α5 expression[J]. Cell Death Differ, 2021,28(1):283-302. DOI: 10.1038/s41418-020-0600-6. [15] Gomez-SanchezJA, PilchKS, van der LansM, et al. After nerve injury, lineage tracing shows that myelin and remak schwann cells elongate extensively and branch to form repair schwann cells, which shorten radically on remyelination[J]. J Neurosci, 2017,37(37):9086-9099. DOI: 10.1523/JNEUROSCI.1453-17.2017. [16] DanovizME, BassanezeV, NakamutaJS, et al. Adipose tissue-derived stem cells from humans and mice differ in proliferative capacity and genome stability in long-term cultures[J]. Stem Cells Dev, 2011,20(4):661-670. DOI: 10.1089/scd.2010.0231. [17] 沈才齐, 李强, 金培生, 等. 人脐带间充质干细胞治疗糖尿病创面效果及其在体内存活、定植研究[J].徐州医科大学学报,2022,42(1):25-29. DOI: 10.3969/j.issn.2096-3882.2022.01.005. [18] Zack-WilliamsSD, ButlerPE, KalaskarDM. Current progress in use of adipose derived stem cells in peripheral nerve regeneration[J]. World J Stem Cells, 2015,7(1):51-64. DOI: 10.4252/wjsc.v7.i1.51. [19] LupuMA, Gradisteanu PircalabioruG, ChifiriucMC, et al. Beneficial effects of food supplements based on hydrolyzed collagen for skin care (Review)[J]. Exp Ther Med, 2020,20(1):12-17. DOI: 10.3892/etm.2019.8342. [20] JiangYH, LouYY, LiTH, et al. Cross-linking methods of type I collagen-based scaffolds for cartilage tissue engineering[J]. Am J Transl Res, 2022,14(2):1146-1159. [21] QinL, GaoH, XiongS, et al. Preparation of collagen/cellulose nanocrystals composite films and their potential applications in corneal repair[J]. J Mater Sci Mater Med, 2020,31(6):55. DOI: 10.1007/s10856-020-06386-6. [22] EvertsP, OnishiK, JayaramP, et al. Platelet-rich plasma: new performance understandings and therapeutic considerations in 2020[J]. Int J Mol Sci, 2020,21(20):7794DOI: 10.3390/ijms21207794. [23] YuW, WangJ, YinJ. Platelet-rich plasma: a promising product for treatment of peripheral nerve regeneration after nerve injury[J]. Int J Neurosci, 2011,121(4):176-180. DOI: 10.3109/00207454.2010.544432. [24] 丁旖, 刘菲, 杨军. 自体富血小板血浆在组织缺损修复中的临床应用进展[J].组织工程与重建外科杂志,2022,18(3):277-280. DOI: 10.3969/j.issn.1673-0364.2022.03.016. [25] 王辉, 韩新生, 焦勇强, 等. 损伤神经周围注射自体PRP对周围神经损伤的修复效果观察[J].卒中与神经疾病,2020,27(2):200-204. DOI: 10.3969/j.issn.1007-0478.2020.02.013. [26] 李立恒, 远洋, 魏建初, 等. 富血小板血浆促进面神经损伤后修复的实验研究[J].中国眼耳鼻喉科杂志,2023,23(2):151-156. DOI: 10.14166/j.issn.1671-2420.2023.02.008. [27] PereiraCT, PaxtonZJ, LiAI. Involvement of PDGF-BB and IGF-1 in activation of human Schwann cells by platelet-rich plasma[J]. Plast Reconstr Surg, 2020,146(6):825e-827e. DOI: 10.1097/PRS.0000000000007406. [28] ZhengC, ZhuQ, LiuX, et al. Effect of platelet-rich plasma (PRP) concentration on proliferation, neurotrophic function and migration of Schwann cells in vitro[J]. J Tissue Eng Regen Med, 2016,10(5):428-436. DOI: 10.1002/term.1756. [29] StolleM, SchulzeJ, RoemerA, et al. Human plasma rich in growth factors improves survival and neurite outgrowth of spiral ganglion neurons in vitro[J]. Tissue Eng Part A, 2018,24(5-6):493-501. DOI: 10.1089/ten.TEA.2017.0120. [30] LienBV, BrownNJ, RansomSC, et al. Enhancing peripheral nerve regeneration with neurotrophic factors and bioengineered scaffolds: a basic science and clinical perspective[J]. J Peripher Nerv Syst, 2020,25(4):320-334. DOI: 10.1111/jns.12414. [31] HendersonCE, PhillipsHS, PollockRA, et al. GDNF: a potent survival factor for motoneurons present in peripheral nerve and muscle[J]. Science, 1994,266(5187):1062-1064. DOI: 10.1126/science.7973664. [32] MarquardtLM, Sakiyama-ElbertSE. GDNF preconditioning can overcome Schwann cell phenotypic memory[J]. Exp Neurol, 2015,265:1-7. DOI: 10.1016/j.expneurol.2014.12.003. [33] JesurajNJ, MarquardtLM, KwasaJA, et al. Glial cell line-derived neurotrophic factor promotes increased phenotypic marker expression in femoral sensory and motor-derived Schwann cell cultures[J]. Exp Neurol, 2014,257:10-18. DOI: 10.1016/j.expneurol.2014.04.005. [34] CaiW, LiuY, ZhangT, et al. GDNF facilitates the differentiation of ADSCs to Schwann cells and enhances nerve regeneration through GDNF/MTA1/Hes1 axis[J]. Arch Biochem Biophys, 2024,753:109893. DOI: 10.1016/j.abb.2024.109893. [35] CaiW, LiuY, ZhangT, et al. GDNF facilitates the differentiation of ADSCs to Schwann cells and enhances nerve regeneration through GDNF/MTA1/Hes1 axis[J]. Arch Biochem Biophys, 2024,753:109893. DOI: 10.1016/j.abb.2024.109893. [36] Cintron-ColonAF, Almeida-AlvesG, VanGyseghemJM, et al. GDNF to the rescue: GDNF delivery effects on motor neurons and nerves, and muscle re-innervation after peripheral nerve injuries[J]. Neural Regen Res, 2022,17(4):748-753. DOI: 10.4103/1673-5374.322446. [37] 杨晨, 胡大海, 郑朝, 等. 过表达胶质细胞源性神经营养因子的脂肪源性间充质干细胞对大鼠电损伤坐骨神经的作用[J].中华烧伤杂志,2015,31(3):199-204. DOI: 10.3760/cma.j.issn.1009-2587.2015.03.010. [38] LeeHL, YeumCE, LeeH, et al. Peripheral nerve-derived stem cell spheroids induce functional recovery and repair after spinal cord injury in rodents[J]. Int J Mol Sci, 2021,22(8)DOI: 10.3390/ijms22084141. -
图 1 原代大鼠脂肪间充质干细胞（ADSC）的鉴定 倒置相差显微镜×40。1A.原代ADSC培养48 h后呈典型的漩涡样排列；1B.第3代ADSC成脂诱导分化14 d后,细胞质内有大量遮光性强的红色脂滴；1C.第3代ADSC成骨诱导分化21 d后,细胞膜表面、细胞质中均可见大量红色或紫红色颗粒状钙化结节
图 2 第3代大鼠脂肪间充质干细胞感染腺病毒48 h后的生长情况 荧光显微镜×40。2A、2B、2C.分别为阴性对照组细胞感染及生长情况的荧光、非荧光、复合图,绝大数细胞感染了腺病毒且生长状态良好；2D、2E、2F.分别为过表达GDNF组细胞感染及生长情况的荧光、非荧光、复合图,绝大数细胞感染了腺病毒,且多数细胞有2个或多个突起（箭头所示）
注：GDNF为胶质细胞源性神经营养因子；阴性对照组、过表达GDNF组细胞分别感染空载腺病毒和过表达GDNF的腺病毒
图 3 富血小板血浆凝胶的微观结构及其中的单纯大鼠脂肪间充质干细胞在培养48 h后的生长情况。3A.凝胶呈三维网状,孔径大小不一 扫描电子显微镜×50；3B.细胞（蓝色箭头指示）可以附着在凝胶中生长 苏木精-伊红染色×20；3C.生长在凝胶中的绝大多数细胞为活细胞（呈绿色）,死细胞（呈红色）很少 钙黄绿素-碘化丙啶×40
图 4 2组感染腺病毒的大鼠脂肪间充质干细胞（ADSC）在富血小板血浆凝胶中培养48 h后的生长情况 ×40。4A.阴性对照凝胶组细胞细胞呈典型的单纯ADSC样梭形生长；4B.过表达GDNF凝胶组的多数细胞伸出2个或多个突起,突起聚集处交织成网状
注：阴性对照凝胶组、过表达胶质细胞源性神经营养因子（GDNF）凝胶组细胞分别感染空载腺病毒和过表达GDNF的腺病毒；成功感染腺病毒的细胞呈绿色
-
蔡维霞.mp4
-
下载:
图(6)
计量
- 文章访问数: 31
- HTML全文浏览量: 12
- PDF下载量: 1
- 被引次数: 0
