应用人脐带血间充质干细胞修复小鼠皮肤创面的实验研究
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摘要
大面积烧伤、创伤、糖尿病、褥疮等原因常造成皮肤正常组织结构及完整性的破坏而形成创面(包括难愈性溃疡)。在无足够自体皮肤进行移植等限制下,如何及时有效的修复创面、重建愈后皮肤正常组织结构以尽可能达到恢复皮肤正常功能的生理性愈合,是目前创面临床治疗的难点。
间充质干细胞(MSC)是来源于中胚层并具有多分化潜能的一类干细胞,广泛的存在于人骨髓、脐带血及胚胎前体组织中,已被应用于组织工程和细胞替代治疗。近年来研究发现,MSC可以跨越胚层界限向皮肤等上皮组织细胞分化,参与组织损伤的修复和功能重建。脐带血间充质干细胞(UCB MSC)较骨髓来源的MSC更接近胚胎干细胞,具有来源更充足、抗原性更低,无创获取等优势,作为一种新型的种子细胞而倍受关注。
本研究中,我们从新生儿脐带血中分离培养MSC,BrdU标记示踪后移植入皮肤缺损创面的SCID小鼠体内。通过检测创面愈合率和皮肤新愈组织内供体细胞的分布及分化情况,探讨了UCB MSC在体内向表皮细胞分化并促进创面修复的可能性,为临床应用人脐带血间充质干细胞修复创面提供理论及实践依据。
材料和方法
一、UCB MSC体外分离培养
应用Ficoll-Hypaque密度梯度离心法从新鲜脐带血中分离MNC,接种于胎牛血清包被过的塑料培养皿中,采用含15%胎牛血清、100U/ml青霉素G、100ug/ml链霉素的DMEM/F12培养基(PH值7.2-7.4),在37℃、5%CO2及饱和湿度环境下培养。过夜即全量换液,弃去未贴壁细胞。此后每3天换液一次。5-7天后在显微镜下挑取出现的MSC克隆,2.5g/L胰酶消化吹散,同前条件传代培养。待细胞生长至80-90%融合时,按1:3比例消化传代培养。
二、流式细胞技术鉴定UCB MSC
消化、收集P7代UCB MSC,应用相关抗体染色后在BD Caliber流式细胞仪检测细胞表面分子CD29、CD34、CD105、HLA-DR的表达。
三、不同代数UCB MSC体外增殖能力的测定
消化收集细胞后以1×103cell/孔接种于24孔板中,3d换液一次,每隔1天随即抽取2孔消化计数、取平均值,连续记录12天。以细胞数为纵轴,时间为横轴,绘制P3、P10代以及BrdU标记的P10代UCB MSC细胞生长曲线,比较不同代数以及BrdU标记后细胞的增殖能力。
四、BrdU标记UCB MSC
将新鲜消化下来的P9代UCB MSC采用含终浓度5μmol/L的BrdU、15%胎牛血清、100U/ml青霉素G、100ug/ml链霉素的DMEM/F12培养基,在37℃、5%CO2及饱和湿度环境下培养。此后3d换液一次,待细胞生长至60%融合时,免疫细胞化学染色检测BrdU阳性细胞率。同时如上检测BrdU标记后对细胞增殖的影响。
五、移植UCB MSC修复SCID小鼠全层皮肤缺损创面
1.创面种植UCB MSC
取4周龄雄性SCID小鼠8只,于小鼠背部中线两侧各制造1×2cm2全层皮肤缺损。消化收集BrdU标记的P9代UCB MSC,以PBS调整细胞悬液密度为2.5×106/ml,将200μl细胞悬液均匀涂布至左侧创面(治疗组),右侧仅涂布PBS(对照组),双侧均以油纱覆盖。于术后7、14天用无菌透明膜描印创面大小,计算比较两组创面愈合率。
2.尾静脉注射移植UCB MSC
按上述方法建立皮肤全层缺损动物模型,设立实验组和对照组各8只。通过尾静脉注射将BrdU标记的P7代UCB MSC移植入实验组SCID小鼠体内,细胞数量为2.5×105/只,对照组注入同体积PBS,创面均以猪皮缝合覆盖。
六、组织学检查
于移植术后第7、14、28天取治疗组和对照组小鼠创周新愈皮肤组织活检,行常规病理检查(HE染色)。在光镜下(×400)观察两组创面新愈组织表皮层厚度以及细胞层数,比较两组创面愈合情况。
七、免疫组织化学染色
于移植术后第7、14、28天取两组创面新愈皮肤组织;于尾静脉注射UCB MSC术后第28天治疗组小鼠远离创面的正常皮肤组织及心、肝等脏器,石蜡包埋切片,SABC法免疫组化染色检测BrdU阳性细胞在上述组织的分布情况。并取尾静脉注射术后不同时相点治疗组创面新愈组织切片,在光镜下(×400)每隔500μm计数一个视野内表皮基底层BrdU阳性细胞数,连续计数3个视野,求其平均值。另行免疫组化双染色检测BrdU阳性细胞及其K19和广谱角蛋白的表达情况,初步探讨UCB MSC在体向人表皮细胞分化的可能机制。
结果
一、UCB MSC体外分离培养、标记及表面标志物的鉴定
优化的分离培养条件传至第3代可以获得较均一的UCB MSC。细胞形态均一,为梭形或成纤维样,呈漩涡样生长。流式检测显示UCB MSC表达CD29(90.30%)、CD105(86.48%),不表达CD34和HLA-DR。免疫细胞化学染色检测BrdU标记P9代UCB MSC标记率约为60%。
二、UCB MSC体外增值特性
UCB MSC体外培养指数生长期倍增时间约为36h,传代接种后1-2 d为潜伏期,从第3天开始细胞进入对数生长期,开始大量增殖。到第9天达到高峰,以后速度减慢,进入平台期。P3、P10代MSC增值速度及生长周期没有明显改变。BrdU标记对UCB MSC的形态、增殖速度及生长周期无明显影响。
三、移植UCB MSC修复小鼠全层皮肤缺损创面
1.创面种植UCB MSC术后治疗组创面愈合率高于对照组(P<0.01)。术后第7d治疗组(56.06±3.04)%,对照组(36.99±2.17)%;术后第14d治疗组(94.75±1.89)%,对照组(84.63±2.28)%。
2.组织学检查表明,治疗组愈合效果优于对照组。比较术后7、14天治疗组和对照组创周新愈皮肤组织表皮厚度及表皮细胞数目显示,治疗组活检组织表皮层明显增厚,细胞及表皮嵴数目显著增多;并在治疗组活检组织中发现有少量皮肤附属器如毛囊形成。
3.免疫组化检测UCB MSC在小鼠体内的分布情况
通过创面直接种植或尾静脉注射两种方式移植UCB MSC,在术后不同时相点的创面新愈组织中都可以检测到来源于脐带血的供体细胞。BrdU染色阳性主要分布于治疗组创面新愈组织的表皮基底层、毛囊内层、棘层以及皮下组织;在形态学上此类细胞呈立方形和圆形,与周围受体本身的表皮细胞无明显差异,可见其相邻生长。比较以上两种移植方式,后者定植于创面的UCB MSC较多。
尾静脉注射移植术后,第7天创面新愈组织表皮基底层BrdU阳性细胞数多于移植后第14天(P<0.01),而移植后第28天与第14天相比表皮基底层BrdU阳性细胞数无明显变化(P>0.05)。术后第28天检测结果显示,在治疗组小鼠远离创面的正常皮肤中仅可见少量随机分布BrdU阳性细胞,其数量远低于创面新愈组织;在治疗组小鼠的肝脏及心脏中也检测到有UCB MSC定植,其数量较少,主要分布于肝小叶中央静脉壁内层和心肌组织中,对照组均为阴性。
四、UCB MSC在小鼠体内向表皮(干)细胞分化情况
免疫组织化学双染色结果显示,尾静脉注射UCB MSC术后第28天在创面新愈组织的表皮基底层、再生皮肤附件毛囊内层,可见部分BrdU阳性细胞同时表达抗K19阳性或抗广谱角蛋白阳性,对照组为阴性;表明部分供体来源的UCB MSC可在创面向表皮(干)细胞分化。
讨论与结论
一、分离所得的MNC接种于胎牛血清包被的培养板后,过夜即全量换液,可以提高MSC克隆形成率,更好的排除破骨样细胞混杂生长的影响,有利于MSC的增殖纯化、提高足月新生儿脐带血MSC的培养成功率。
二、创面应用UCB MSC移植后,可以显著提高创面愈合率,改善创面愈合效果。治疗组创面更接近生理性愈合的效果,其新愈皮肤组织的表皮层厚度增加、细胞层数增多;且证实有部分供体细胞参与了创面再生皮肤附件的形成。通过免疫组化双染色结果,初步推断其机制为皮肤损伤条件下的创面微环境可以诱导UCB MSC在小鼠体内向表皮(干)细胞分化。移植UCB MSC可以通过补充创面修复细胞的数量参与创面的修复和重建,可以促进创面愈合速度,改善创面愈合质量。
三、尾静脉注射移植UCB MSC术后治疗组小鼠远离创面的正常皮肤中,只有少数供体细胞随机分布;且随着创面的愈合,供体细胞定植于创面的细胞数量有减少趋势,但在愈合后期,这种趋势不再继续。实验表明创面的存在对UCB MSC有较强的趋化作用,有利于更多的MSC参与创面修复。
Cutaneous wounds feature the destruction of the integrity of the normal skin tissue. Because of the lack of the auto-skin, it is soundly difficult for the clinical therapy in efficaciously and quickly reparing the wounds and reconstructing the normal skin tissue for the sake of physiological agglutination of the normal skin function.
Mesenchymal stem cells, widly exist in human bone marrow, umbilical cord blood and embryo-precurssor, are originated from mesoderm progenitor and capable of multipotential differentiation, which is thus applied in tissue engineering and cell-replacement therapeutics. The recent study demonstrates that MSCs could also differentiate into epithelium, such as skin, and participate in regenerating and repairing the injuries. UCB is considered as a new alternative sources for MSCs. Compared with that from BM, there are many advantages,such as lower antigenicity, more sufficient source and noninvasive aqcuiring.
In our study, MSCs was isolated from the neonate UCB and expanded in vitro. After the cells were labled with 5-bromodeoxyuridine (BrdU), and then adoptively transferred into SCID mice. We addressed the possibility that the UCB-derived MSCs differentiated into epidermic cells and facilitated the wound restoration by detecting the wound consolidation ratio and elucidating the distribution and differentiation status of the donor cells in the newly concrescent skin, which might theoretically and pratically support the clinical application of human umbilical cord blood in wound restoration.
Materials and methods:
1. Isolation and expansion of MSCs from UCB
The mononuclear cells (MNC) were isolated from the fresh full-term UCB by density gradient centrifugation with Ficoll-Hypaque. The cells were innoculated into the plastic culture flask coated by the FBS and cultured overnight in DMEM/F12 medium supplemented with 15% FBS and 100U/ml Peni-Strep at 37℃、5%CO2 and saturated humidity. The medium was changed completely, to remove the unattached cells. After that, the medium was refreshed every 3 days. Usually after 5-7 days, some cell clones could be found and picked up under microscope. The clones were trypsinizated and cultured in the same condition mentioned above. When the cells grew to 80%-90% confluence, they should be passaged in the ratio of 1:3.
2. Identification of the UCB-derived MSCs via flow cytometry
The expanded cells at passage 7 were stained with PE- or FITC-labelled CD29, CD34, CD105 or HLA-DR after, respectively, and the expressions of relative molecules were checked by flow cytometry.
3. Proliferative potency of isolated MSCs from UCB
The cultured cell at passage 3 and passage 10 were seeded into the 24-well plates (1×103cell/well) respectively. The medium was changed every 3 days. The cells from two wells were harvested and counted every 2 days till to day 12 after inoculateion. The cell populations were set into longitudinal axis, the timepoints were put into abscissa axis.
4. BrdU labelling of UCB-derived MSCs and the detection of the labelling rate
The fresh MSC from UCB at passage 9 were cultured under the same condition with BrdU at the final concentration of 5μmol/L. The medium was changed every 3 days as above. When the cells grew to about 60% confluence, the efficiency of BrdU labeling were assayed by immunocytochemistry. Meanwhile, the proliferateion potencies of the BrdU labeled cells were checked as above.
5. Application of cultured MSCs from UCB to repair the full thickness skin defect wound in SCID mice
1) Implantion of UCB-derived MSCs directly on the wound
SCID mice, male, 4-week-old, were prepared two the full thickness skin defect wounds (1×2cm2) on the both sides of posterolateral thorax. The BrdU labeled MSCs from UCB were collected and washed with PBS, and 5×105 cells in 0.2ml were implanted onto the left wounds of the mice, while PBS was smeared onto the right wound as control. Both of the sides were coverd by vaslin dressing. The size of the remained wounds was measured by asepsis fenestrate membrane on the day 7 and 14 after the operation and the healing rate was caculated by the correlated formula.
2) Injection of UCB-derived MSCs into mice via tail vein
After the full thickness skin defect wounds on the back of SCID mice was made as above, about 2.5×105 BrdU labeled MSCs from UCB in 0.1ml volume were injected into every mouse via tail vein immediately one by one. PBS was injected into the control group. The wounds were covered with pigskin. There were 8 mice in each group.
6. Histological examination
On the 7th, 14th and 28th day after transplantation, regenerated skin tissue were biopsied from the experimental and control group, The sample were embedded with paraffin and sectioned after fixed with formalin solution. Hematoxylin-eosin (HE) staining and histological observation was according to the standard method.
7. Immunohistochemical staining
Newly regenerated skin tissue was biopsied on the 7th, 14th and 28th day after transplantation from the two groups, respectively. The normal skin tissue far away from the wounds, and some internal organs such as heart and liver, were harvested on the 28th day after injecting UCB-derived MSCs via tail vein. All the samples were sectioned as above. The distribution and number of BrdU positive cells were observed and counted after immunohistochemistry (SABC) staining. As far as the section of skin tissue sample, the double positive cells stained with anti-BrdU antibody and anti-Keratin 19 antibody were detected.
Results:
1. MSCs were isolated from UCB successfully.
With the method described above, some clones could be found after the MNCs isolated from UCB were cultured for about 7 to 14 days. The cells picked from the clones could be passaged, and grew like whirlpool as the same morphous as fusiform shape or similar to the fibroblast. It was found by FACS that the cells at 3rd generation could express CD29() and CD105, the positive rates were90.30% and 86.48% respectively, while neither CD34 nor HLA-DR were detectable on the cells.
2. BrdU could label the isolated MSC from UCB.
It was found the the positive rate of BrdU labeled cells was about 60% by flow cytometry, and the labeled cells grew and expanded as same as non-labeled cells.
3. The proliferation characteristics of UCB-derived MSCs in vitro
The study found that the eclipse period of UCB-derived MSCs in vitro was 1-2 days after passage. The cells grew into the exponential phase since the 3rd day, and they proliferated actively and got to the peak on the 9th day. After that, the speed of growth slew down and the proliferation entered into the platform stage. There was no significant difference between the MSC proliferative speed and the growth cycle in the various generations, i.e. P3 and P10.
4. Application of UCB-derived MSCs to repairing the full thickness defect
1). The healing rate was apparently higher in the UCB-derived MSCs implanting group than that in the control(P<0.01), which is (56.06±3.04)% vs (36.99±2.17)% on the 7th day postoperative and (94.75±1.89)% vs (84.63±2.28)% on the 14th day after operation.
2). The histological examination elucidated that the wound healing quality in the experimental group looked better than those in the control. In the comparison of the epidermisc layer thickness and the amount of the cells from the newly regenerated skin tissue on the 7th and the 14th day after operation, we found that the epidermisc layer was obviously thicker and the amount of the cells and the dermal ridges was much more in the experimental group than those in the control. Additionally, skin appendages, such as folliculus pili, emerged in the experimental group.
3). The distribution of the UCB-derived MSCs in vivo via immunohistochemistry
We were able to detect donor cells derived from umbilical cord blood in the newly regenetated skin tissue at the different time points after operation by UCB-derived MSCs were either implanted on the wound or injected into the mice via tail vein. The BrdU positive cells were found in the basal layer of epidermis, inner layer of folliculus pili, spinous layer and superficial fascia in the newly regenetated skin tissue in the expeimental group. The BrdU positive cells were cube or spherical in the shape and located close to the recipient cells.
Furthermore, it was found that there were more the BrdU positive cells located in the basal layer of epidermis in the newly regenerated skin tissue on the 7th day than that on the 14th day after the donor cells injected via tail vein(P<0.01),while there was no significant difference on the 28th day compared with that on the 14th day. Moreover, there were small amount of BrdU positive cells randomly distributing in the normal skin tissue far away from the wound, which was overwhelmingly less than that in the newly regenerated skin tissue. There were also small quantity of UCB-derived MSCs located in the hearts and the livers of the experimental group and mainly existed in the inner-wall of central veins of hepatic lobules and cardiac muscular tissues.
5. UCB-derived MSCs differentiation to epidermic (stem) cells in vivo
It was found that there were BrdU positive cells in the basal layer of epidermis and the inner layer of folliculus pili from the newly regenerated skin tissue on the 28th after injecting UCB-derived MSCs via tail vein, and some of them simultaneouslly expressed keratin 19, which meant that some UCB-derived MSCs of the donor might be able to differentiate to epidermic (stem) cells in vivo.
Discussion and conclusion
1. The protocol that the isolated MNC were innoculated into the culture flask coated by the FBS overnight and the medium was completely refreshed was able to increase the cloning efficiency of MSC, which could eliminate the influence of the osteoclast, facilitate the purification and proliferation of MSC. Finally, MSC derived from the umbilical cord blood of the full-term neonate could be easy and successful obtainment.
2. Implantion of UCB-derived MSCs on the wound is able to significantly increase the healing rate and improve the healing quality. The healing wounds in the experimental group looked more similar to the physiological status that the thickness and the cell population of the cuticular layer increase in the newly regenerated skin tissue. Additionlly, it is proved that some donor cells are involved in the formation of newly regenerated appendages of the skin
It can be initially concluded that the UCB-derived MSCs can be induced to epidermic (stem) cells by the microenviroment in the condition of skin injury and the transplantation of UCB-derived MSCs may participate in the repair and reestablishment of the wound by supplementing the repair cell population.
3. Very few MSCs were found randomly in the normal skin far away from the wound after injecting via tail vein, while there were a lot of MSC distributing in or around the wound, which indicated that the wound itself could have the chemotaxis to the MSC
间充质干细胞(MSC)是来源于中胚层并具有多分化潜能的一类干细胞,广泛的存在于人骨髓、脐带血及胚胎前体组织中,已被应用于组织工程和细胞替代治疗。近年来研究发现,MSC可以跨越胚层界限向皮肤等上皮组织细胞分化,参与组织损伤的修复和功能重建。脐带血间充质干细胞(UCB MSC)较骨髓来源的MSC更接近胚胎干细胞,具有来源更充足、抗原性更低,无创获取等优势,作为一种新型的种子细胞而倍受关注。
本研究中,我们从新生儿脐带血中分离培养MSC,BrdU标记示踪后移植入皮肤缺损创面的SCID小鼠体内。通过检测创面愈合率和皮肤新愈组织内供体细胞的分布及分化情况,探讨了UCB MSC在体内向表皮细胞分化并促进创面修复的可能性,为临床应用人脐带血间充质干细胞修复创面提供理论及实践依据。
材料和方法
一、UCB MSC体外分离培养
应用Ficoll-Hypaque密度梯度离心法从新鲜脐带血中分离MNC,接种于胎牛血清包被过的塑料培养皿中,采用含15%胎牛血清、100U/ml青霉素G、100ug/ml链霉素的DMEM/F12培养基(PH值7.2-7.4),在37℃、5%CO2及饱和湿度环境下培养。过夜即全量换液,弃去未贴壁细胞。此后每3天换液一次。5-7天后在显微镜下挑取出现的MSC克隆,2.5g/L胰酶消化吹散,同前条件传代培养。待细胞生长至80-90%融合时,按1:3比例消化传代培养。
二、流式细胞技术鉴定UCB MSC
消化、收集P7代UCB MSC,应用相关抗体染色后在BD Caliber流式细胞仪检测细胞表面分子CD29、CD34、CD105、HLA-DR的表达。
三、不同代数UCB MSC体外增殖能力的测定
消化收集细胞后以1×103cell/孔接种于24孔板中,3d换液一次,每隔1天随即抽取2孔消化计数、取平均值,连续记录12天。以细胞数为纵轴,时间为横轴,绘制P3、P10代以及BrdU标记的P10代UCB MSC细胞生长曲线,比较不同代数以及BrdU标记后细胞的增殖能力。
四、BrdU标记UCB MSC
将新鲜消化下来的P9代UCB MSC采用含终浓度5μmol/L的BrdU、15%胎牛血清、100U/ml青霉素G、100ug/ml链霉素的DMEM/F12培养基,在37℃、5%CO2及饱和湿度环境下培养。此后3d换液一次,待细胞生长至60%融合时,免疫细胞化学染色检测BrdU阳性细胞率。同时如上检测BrdU标记后对细胞增殖的影响。
五、移植UCB MSC修复SCID小鼠全层皮肤缺损创面
1.创面种植UCB MSC
取4周龄雄性SCID小鼠8只,于小鼠背部中线两侧各制造1×2cm2全层皮肤缺损。消化收集BrdU标记的P9代UCB MSC,以PBS调整细胞悬液密度为2.5×106/ml,将200μl细胞悬液均匀涂布至左侧创面(治疗组),右侧仅涂布PBS(对照组),双侧均以油纱覆盖。于术后7、14天用无菌透明膜描印创面大小,计算比较两组创面愈合率。
2.尾静脉注射移植UCB MSC
按上述方法建立皮肤全层缺损动物模型,设立实验组和对照组各8只。通过尾静脉注射将BrdU标记的P7代UCB MSC移植入实验组SCID小鼠体内,细胞数量为2.5×105/只,对照组注入同体积PBS,创面均以猪皮缝合覆盖。
六、组织学检查
于移植术后第7、14、28天取治疗组和对照组小鼠创周新愈皮肤组织活检,行常规病理检查(HE染色)。在光镜下(×400)观察两组创面新愈组织表皮层厚度以及细胞层数,比较两组创面愈合情况。
七、免疫组织化学染色
于移植术后第7、14、28天取两组创面新愈皮肤组织;于尾静脉注射UCB MSC术后第28天治疗组小鼠远离创面的正常皮肤组织及心、肝等脏器,石蜡包埋切片,SABC法免疫组化染色检测BrdU阳性细胞在上述组织的分布情况。并取尾静脉注射术后不同时相点治疗组创面新愈组织切片,在光镜下(×400)每隔500μm计数一个视野内表皮基底层BrdU阳性细胞数,连续计数3个视野,求其平均值。另行免疫组化双染色检测BrdU阳性细胞及其K19和广谱角蛋白的表达情况,初步探讨UCB MSC在体向人表皮细胞分化的可能机制。
结果
一、UCB MSC体外分离培养、标记及表面标志物的鉴定
优化的分离培养条件传至第3代可以获得较均一的UCB MSC。细胞形态均一,为梭形或成纤维样,呈漩涡样生长。流式检测显示UCB MSC表达CD29(90.30%)、CD105(86.48%),不表达CD34和HLA-DR。免疫细胞化学染色检测BrdU标记P9代UCB MSC标记率约为60%。
二、UCB MSC体外增值特性
UCB MSC体外培养指数生长期倍增时间约为36h,传代接种后1-2 d为潜伏期,从第3天开始细胞进入对数生长期,开始大量增殖。到第9天达到高峰,以后速度减慢,进入平台期。P3、P10代MSC增值速度及生长周期没有明显改变。BrdU标记对UCB MSC的形态、增殖速度及生长周期无明显影响。
三、移植UCB MSC修复小鼠全层皮肤缺损创面
1.创面种植UCB MSC术后治疗组创面愈合率高于对照组(P<0.01)。术后第7d治疗组(56.06±3.04)%,对照组(36.99±2.17)%;术后第14d治疗组(94.75±1.89)%,对照组(84.63±2.28)%。
2.组织学检查表明,治疗组愈合效果优于对照组。比较术后7、14天治疗组和对照组创周新愈皮肤组织表皮厚度及表皮细胞数目显示,治疗组活检组织表皮层明显增厚,细胞及表皮嵴数目显著增多;并在治疗组活检组织中发现有少量皮肤附属器如毛囊形成。
3.免疫组化检测UCB MSC在小鼠体内的分布情况
通过创面直接种植或尾静脉注射两种方式移植UCB MSC,在术后不同时相点的创面新愈组织中都可以检测到来源于脐带血的供体细胞。BrdU染色阳性主要分布于治疗组创面新愈组织的表皮基底层、毛囊内层、棘层以及皮下组织;在形态学上此类细胞呈立方形和圆形,与周围受体本身的表皮细胞无明显差异,可见其相邻生长。比较以上两种移植方式,后者定植于创面的UCB MSC较多。
尾静脉注射移植术后,第7天创面新愈组织表皮基底层BrdU阳性细胞数多于移植后第14天(P<0.01),而移植后第28天与第14天相比表皮基底层BrdU阳性细胞数无明显变化(P>0.05)。术后第28天检测结果显示,在治疗组小鼠远离创面的正常皮肤中仅可见少量随机分布BrdU阳性细胞,其数量远低于创面新愈组织;在治疗组小鼠的肝脏及心脏中也检测到有UCB MSC定植,其数量较少,主要分布于肝小叶中央静脉壁内层和心肌组织中,对照组均为阴性。
四、UCB MSC在小鼠体内向表皮(干)细胞分化情况
免疫组织化学双染色结果显示,尾静脉注射UCB MSC术后第28天在创面新愈组织的表皮基底层、再生皮肤附件毛囊内层,可见部分BrdU阳性细胞同时表达抗K19阳性或抗广谱角蛋白阳性,对照组为阴性;表明部分供体来源的UCB MSC可在创面向表皮(干)细胞分化。
讨论与结论
一、分离所得的MNC接种于胎牛血清包被的培养板后,过夜即全量换液,可以提高MSC克隆形成率,更好的排除破骨样细胞混杂生长的影响,有利于MSC的增殖纯化、提高足月新生儿脐带血MSC的培养成功率。
二、创面应用UCB MSC移植后,可以显著提高创面愈合率,改善创面愈合效果。治疗组创面更接近生理性愈合的效果,其新愈皮肤组织的表皮层厚度增加、细胞层数增多;且证实有部分供体细胞参与了创面再生皮肤附件的形成。通过免疫组化双染色结果,初步推断其机制为皮肤损伤条件下的创面微环境可以诱导UCB MSC在小鼠体内向表皮(干)细胞分化。移植UCB MSC可以通过补充创面修复细胞的数量参与创面的修复和重建,可以促进创面愈合速度,改善创面愈合质量。
三、尾静脉注射移植UCB MSC术后治疗组小鼠远离创面的正常皮肤中,只有少数供体细胞随机分布;且随着创面的愈合,供体细胞定植于创面的细胞数量有减少趋势,但在愈合后期,这种趋势不再继续。实验表明创面的存在对UCB MSC有较强的趋化作用,有利于更多的MSC参与创面修复。
Cutaneous wounds feature the destruction of the integrity of the normal skin tissue. Because of the lack of the auto-skin, it is soundly difficult for the clinical therapy in efficaciously and quickly reparing the wounds and reconstructing the normal skin tissue for the sake of physiological agglutination of the normal skin function.
Mesenchymal stem cells, widly exist in human bone marrow, umbilical cord blood and embryo-precurssor, are originated from mesoderm progenitor and capable of multipotential differentiation, which is thus applied in tissue engineering and cell-replacement therapeutics. The recent study demonstrates that MSCs could also differentiate into epithelium, such as skin, and participate in regenerating and repairing the injuries. UCB is considered as a new alternative sources for MSCs. Compared with that from BM, there are many advantages,such as lower antigenicity, more sufficient source and noninvasive aqcuiring.
In our study, MSCs was isolated from the neonate UCB and expanded in vitro. After the cells were labled with 5-bromodeoxyuridine (BrdU), and then adoptively transferred into SCID mice. We addressed the possibility that the UCB-derived MSCs differentiated into epidermic cells and facilitated the wound restoration by detecting the wound consolidation ratio and elucidating the distribution and differentiation status of the donor cells in the newly concrescent skin, which might theoretically and pratically support the clinical application of human umbilical cord blood in wound restoration.
Materials and methods:
1. Isolation and expansion of MSCs from UCB
The mononuclear cells (MNC) were isolated from the fresh full-term UCB by density gradient centrifugation with Ficoll-Hypaque. The cells were innoculated into the plastic culture flask coated by the FBS and cultured overnight in DMEM/F12 medium supplemented with 15% FBS and 100U/ml Peni-Strep at 37℃、5%CO2 and saturated humidity. The medium was changed completely, to remove the unattached cells. After that, the medium was refreshed every 3 days. Usually after 5-7 days, some cell clones could be found and picked up under microscope. The clones were trypsinizated and cultured in the same condition mentioned above. When the cells grew to 80%-90% confluence, they should be passaged in the ratio of 1:3.
2. Identification of the UCB-derived MSCs via flow cytometry
The expanded cells at passage 7 were stained with PE- or FITC-labelled CD29, CD34, CD105 or HLA-DR after, respectively, and the expressions of relative molecules were checked by flow cytometry.
3. Proliferative potency of isolated MSCs from UCB
The cultured cell at passage 3 and passage 10 were seeded into the 24-well plates (1×103cell/well) respectively. The medium was changed every 3 days. The cells from two wells were harvested and counted every 2 days till to day 12 after inoculateion. The cell populations were set into longitudinal axis, the timepoints were put into abscissa axis.
4. BrdU labelling of UCB-derived MSCs and the detection of the labelling rate
The fresh MSC from UCB at passage 9 were cultured under the same condition with BrdU at the final concentration of 5μmol/L. The medium was changed every 3 days as above. When the cells grew to about 60% confluence, the efficiency of BrdU labeling were assayed by immunocytochemistry. Meanwhile, the proliferateion potencies of the BrdU labeled cells were checked as above.
5. Application of cultured MSCs from UCB to repair the full thickness skin defect wound in SCID mice
1) Implantion of UCB-derived MSCs directly on the wound
SCID mice, male, 4-week-old, were prepared two the full thickness skin defect wounds (1×2cm2) on the both sides of posterolateral thorax. The BrdU labeled MSCs from UCB were collected and washed with PBS, and 5×105 cells in 0.2ml were implanted onto the left wounds of the mice, while PBS was smeared onto the right wound as control. Both of the sides were coverd by vaslin dressing. The size of the remained wounds was measured by asepsis fenestrate membrane on the day 7 and 14 after the operation and the healing rate was caculated by the correlated formula.
2) Injection of UCB-derived MSCs into mice via tail vein
After the full thickness skin defect wounds on the back of SCID mice was made as above, about 2.5×105 BrdU labeled MSCs from UCB in 0.1ml volume were injected into every mouse via tail vein immediately one by one. PBS was injected into the control group. The wounds were covered with pigskin. There were 8 mice in each group.
6. Histological examination
On the 7th, 14th and 28th day after transplantation, regenerated skin tissue were biopsied from the experimental and control group, The sample were embedded with paraffin and sectioned after fixed with formalin solution. Hematoxylin-eosin (HE) staining and histological observation was according to the standard method.
7. Immunohistochemical staining
Newly regenerated skin tissue was biopsied on the 7th, 14th and 28th day after transplantation from the two groups, respectively. The normal skin tissue far away from the wounds, and some internal organs such as heart and liver, were harvested on the 28th day after injecting UCB-derived MSCs via tail vein. All the samples were sectioned as above. The distribution and number of BrdU positive cells were observed and counted after immunohistochemistry (SABC) staining. As far as the section of skin tissue sample, the double positive cells stained with anti-BrdU antibody and anti-Keratin 19 antibody were detected.
Results:
1. MSCs were isolated from UCB successfully.
With the method described above, some clones could be found after the MNCs isolated from UCB were cultured for about 7 to 14 days. The cells picked from the clones could be passaged, and grew like whirlpool as the same morphous as fusiform shape or similar to the fibroblast. It was found by FACS that the cells at 3rd generation could express CD29() and CD105, the positive rates were90.30% and 86.48% respectively, while neither CD34 nor HLA-DR were detectable on the cells.
2. BrdU could label the isolated MSC from UCB.
It was found the the positive rate of BrdU labeled cells was about 60% by flow cytometry, and the labeled cells grew and expanded as same as non-labeled cells.
3. The proliferation characteristics of UCB-derived MSCs in vitro
The study found that the eclipse period of UCB-derived MSCs in vitro was 1-2 days after passage. The cells grew into the exponential phase since the 3rd day, and they proliferated actively and got to the peak on the 9th day. After that, the speed of growth slew down and the proliferation entered into the platform stage. There was no significant difference between the MSC proliferative speed and the growth cycle in the various generations, i.e. P3 and P10.
4. Application of UCB-derived MSCs to repairing the full thickness defect
1). The healing rate was apparently higher in the UCB-derived MSCs implanting group than that in the control(P<0.01), which is (56.06±3.04)% vs (36.99±2.17)% on the 7th day postoperative and (94.75±1.89)% vs (84.63±2.28)% on the 14th day after operation.
2). The histological examination elucidated that the wound healing quality in the experimental group looked better than those in the control. In the comparison of the epidermisc layer thickness and the amount of the cells from the newly regenerated skin tissue on the 7th and the 14th day after operation, we found that the epidermisc layer was obviously thicker and the amount of the cells and the dermal ridges was much more in the experimental group than those in the control. Additionally, skin appendages, such as folliculus pili, emerged in the experimental group.
3). The distribution of the UCB-derived MSCs in vivo via immunohistochemistry
We were able to detect donor cells derived from umbilical cord blood in the newly regenetated skin tissue at the different time points after operation by UCB-derived MSCs were either implanted on the wound or injected into the mice via tail vein. The BrdU positive cells were found in the basal layer of epidermis, inner layer of folliculus pili, spinous layer and superficial fascia in the newly regenetated skin tissue in the expeimental group. The BrdU positive cells were cube or spherical in the shape and located close to the recipient cells.
Furthermore, it was found that there were more the BrdU positive cells located in the basal layer of epidermis in the newly regenerated skin tissue on the 7th day than that on the 14th day after the donor cells injected via tail vein(P<0.01),while there was no significant difference on the 28th day compared with that on the 14th day. Moreover, there were small amount of BrdU positive cells randomly distributing in the normal skin tissue far away from the wound, which was overwhelmingly less than that in the newly regenerated skin tissue. There were also small quantity of UCB-derived MSCs located in the hearts and the livers of the experimental group and mainly existed in the inner-wall of central veins of hepatic lobules and cardiac muscular tissues.
5. UCB-derived MSCs differentiation to epidermic (stem) cells in vivo
It was found that there were BrdU positive cells in the basal layer of epidermis and the inner layer of folliculus pili from the newly regenerated skin tissue on the 28th after injecting UCB-derived MSCs via tail vein, and some of them simultaneouslly expressed keratin 19, which meant that some UCB-derived MSCs of the donor might be able to differentiate to epidermic (stem) cells in vivo.
Discussion and conclusion
1. The protocol that the isolated MNC were innoculated into the culture flask coated by the FBS overnight and the medium was completely refreshed was able to increase the cloning efficiency of MSC, which could eliminate the influence of the osteoclast, facilitate the purification and proliferation of MSC. Finally, MSC derived from the umbilical cord blood of the full-term neonate could be easy and successful obtainment.
2. Implantion of UCB-derived MSCs on the wound is able to significantly increase the healing rate and improve the healing quality. The healing wounds in the experimental group looked more similar to the physiological status that the thickness and the cell population of the cuticular layer increase in the newly regenerated skin tissue. Additionlly, it is proved that some donor cells are involved in the formation of newly regenerated appendages of the skin
It can be initially concluded that the UCB-derived MSCs can be induced to epidermic (stem) cells by the microenviroment in the condition of skin injury and the transplantation of UCB-derived MSCs may participate in the repair and reestablishment of the wound by supplementing the repair cell population.
3. Very few MSCs were found randomly in the normal skin far away from the wound after injecting via tail vein, while there were a lot of MSC distributing in or around the wound, which indicated that the wound itself could have the chemotaxis to the MSC
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2. Robert JD,Annemarie BM. Mesenchymal stem cells:Biology and potential clinical uses. Experimental Hematology. 2000;28:875-884
3. David JA, Fred HG, Irving LW. Can stem cells cross the lineage boundaries. Nature Medicine. 2001;7(4):393-395
4. Badiavas EV,Falanga V. Treatment of chronic wounds with bone marrow-derived cells. Arch Dermatol 2003;139:510-516.
5. Sueblinvong V, Loi R, Eisenhauer PL, et al. Derivation of lung epithelium from human cord blood-derived mesenchymal stem cells. Am J Respir Crit Care Med,2007.
6.付小兵,方利君,王立新,等.髓髓间充质干细胞自体移植促进猪皮肤损伤的修复质量.中华医学杂志,2004,84(11):920-924.
7.呼和塔娜,郭丽,凌翎,等.骨髓间充质干细胞移植对豚鼠皮肤深Ⅱ度烧伤的治疗作用.中国康复理论与实践,2005,11(1):13-15
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