人脐带间充质干细胞联合自体Meek微型皮片移植在大面积烧伤患者中应用的前瞻性研究

闫甜甜; 肖荣; 王影; 林国安; 郑银; 赵辉; 李文军; 尚新志; 孟进松; 胡东升; 李嵩; 王超; 林之琛; 陈鸿昌; 赵东彦; 唐棣

doi:10.3760/cma.j.cn501225-20220728-00319

人脐带间充质干细胞联合自体Meek微型皮片移植在大面积烧伤患者中应用的前瞻性研究

doi: 10.3760/cma.j.cn501225-20220728-00319

闫甜甜¹,
肖荣^1, ,,
王影^2, ,,
林国安¹,
郑银¹,
赵辉³,
李文军¹,
尚新志¹,
孟进松¹,
胡东升¹,
李嵩¹,
王超¹,
林之琛¹,
陈鸿昌¹,
赵东彦¹,
唐棣⁴

1.
解放军联勤保障部队第九九〇医院全军烧伤中心，驻马店　　463002
2.
北京中京高科生物科技有限公司，北京　　100089
3.
河南省细胞产业技术研究院有限公司，郑州　　450121
4.
解放军中部战区总医院烧伤整形科，武汉　　430012

基金项目:

军队医学科技青年培育计划项目 20QNPY074

详细信息

通讯作者:
肖荣，Email：xr131@hotmail.com

王影，Email：2669970157@qq.com

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出版历程
- 收稿日期: 2022-07-28

A prospective study on application of human umbilical cord mesenchymal stem cells combined with autologous Meek microskin transplantation in patients with extensive burns

1.
Military Burn Center, the 990th Hospital of PLA Joint Logistics Support Force, Zhumadian 463002, China
2.
Beijing Zhongjing Hi-Tech Biotechnology Co., Beijing 100089, China
3.
Henan Cellular Industry Technology Research Institute Co., Zhengzhou 450121, China
4.
Department of BurnsPlastic Surgery, PLA General Hospital of Central Theater Command, Wuhan 430012, China

Funds:

Military Medical Science and Technology Youth Incubation Program 20QNPY074

More Information

Corresponding author: Xiao Rong, Email: xr131@hotmail.com; Wang Ying, Email: 2669970157@qq.com

摘要

摘要: 目的探讨人脐带间充质干细胞（hUCMSC）联合自体Meek微型皮片移植对大面积烧伤患者的效果。方法采用前瞻性自身对照研究方法。2019年5月—2022年6月，解放军联勤保障部队第九九〇医院收治的16例大面积烧伤患者符合入选标准，按照剔除标准剔除3例患者，最终纳入13例患者，其中男10例、女3例，年龄24~61（42±13）岁。共选择受试区20个（创面40个，大小均为10 cm×10 cm）。采用随机数字表法将每个受试区中的2个相邻创面分为涂抹含hUCMSC透明质酸凝胶的hUCMSC+凝胶组及涂抹单纯透明质酸凝胶的单纯凝胶组，每组20个创面，随后2组创面均移植扩展比例为1∶6的自体Meek微型皮片。术后2、3、4周，观察创面愈合情况并计算创面愈合率，记录创面愈合时间。术后，若创面出现脓性分泌物，则采集创面分泌物标本行微生物培养。术后3、6、12个月，采用温哥华瘢痕量表评估创面瘢痕增生情况。术后3个月，取创面组织，行苏木精-伊红（HE）染色观察皮肤组织形态学变化，行免疫组织化学染色观察Ki67阳性和波形蛋白阳性表达情况并统计阳性细胞数。对数据行配对样本t检验及Bonferroni校正。结果术后2、3、4周，hUCMSC+凝胶组创面愈合率分别为（80±11）%、（84±12）%、（92±9）%，均明显高于单纯凝胶组的（67±18）%、（74±21）%、（84±16）%（t值分别为4.01、3.52、3.66，P<0.05）。hUCMSC+凝胶组创面愈合时间为（31±11）d，较单纯凝胶组的（36±13）d明显缩短（t=-3.68，P<0.05）。术后，相邻的2组创面分泌物标本微生物培养情况均相同，4个受试区结果为阴性，16个受试区结果为阳性。术后3、6、12个月，单纯凝胶组创面温哥华瘢痕量表评分分别为（7.8±1.9）、（6.7±2.1）、（5.4±1.6）分，均明显高于hUCMSC+凝胶组的（6.8±1.8）、（5.6±1.6）、（4.0±1.4）分（t值分别为-4.79、-4.37、-5.47，P<0.05）。术后3个月，HE染色显示，hUCMSC+凝胶组创面表皮层厚度、表皮嵴等较单纯凝胶组增加；免疫组织化学染色显示，hUCMSC+凝胶组创面Ki67阳性细胞数较单纯凝胶组明显增加（t=4.39，P<0.05），2组创面波形蛋白阳性细胞数无明显差异（P>0.05）。结论将含hUCMSC的透明质酸凝胶涂抹于创面的应用方式操作方便，是一种较佳的给药途径。hUCMSC外用可促进大面积烧伤患者自体Meek微型皮片移植术区愈合，缩短创面愈合时间，减轻瘢痕增生，可能与创面表皮层厚度、表皮嵴增加和细胞增殖活跃有关。
- 烧伤 /
- 瘢痕 /
- 间质干细胞移植 /
- 皮肤移植 /
- Meek微型皮片
Abstract: Objective To investigate the effects of human umbilical cord mesenchymal stem cells (hUCMSCs) combined with autologous Meek microskin transplantation on patients with extensive burns. Methods The prospective self-controlled study was conducted. From May 2019 to June 2022, 16 patients with extensive burns admitted to the 990^th Hospital of PLA Joint Logistics Support Force met the inclusion criteria, while 3 patients were excluded according to the exclusion criteria, and 13 patients were finally selected, including 10 males and 3 females, aged 24-61 (42±13) years. A total of 20 trial areas (40 wounds, with area of 10 cm×10 cm in each wound) were selected. Two adjacent wounds in each trial area were divided into hUCMSC+gel group applied with hyaluronic acid gel containing hUCMSCs and gel only group applied with hyaluronic acid gel only according to the random number table, with 20 wounds in each group. Afterwards the wounds in two groups were transplanted with autologous Meek microskin grafts with an extension ratio of 1∶6. In 2, 3, and 4 weeks post operation, the wound healing was observed, the wound healing rate was calculated, and the wound healing time was recorded. The specimen of wound secretion was collected for microorganism culture if there was purulent secretion on the wound post operation. In 3, 6, and 12 months post operation, the scar hyperplasia in wound was assessed using the Vancouver scar scale (VSS). In 3 months post operation, the wound tissue was collected for hematoxylin-eosin (HE) staining to observe the morphological changes and for immunohistochemical staining to observe the positive expressions of Ki67 and vimentin and to count the number of positive cells. Data were statistically analyzed with paired samples t test and Bonferronni correction. Results In 2, 3, and 4 weeks post operation, the wound healing rates in hUCMSC+gel group were (80±11)%, (84±12)%, and (92±9)%, respectively, which were significantly higher than (67±18)%, (74±21)%, and (84±16)% in gel only group (with t values of 4.01, 3.52, and 3.66, respectively, P<0.05). The wound healing time in hUCMSC+gel group was (31±11) d, which was significantly shorter than (36±13) d in gel only group (t=-3.68, P<0.05). The microbiological culture of the postoperative wound secretion specimens from the adjacent wounds in 2 groups was identical, with negative results in 4 trial areas and positive results in 16 trial areas. In 3, 6, and 12 months post operation, the VSS scores of wounds in gel only group were 7.8±1.9, 6.7±2.1, and 5.4±1.6, which were significantly higher than 6.8±1.8, 5.6±1.6, and 4.0±1.4 in hUCMSC+gel group, respectively (with t values of -4.79, -4.37, and -5.47, respectively, P<0.05). In 3 months post operation, HE staining showed an increase in epidermal layer thickness and epidermal crest in wound in hUCMSC+gel group compared with those in gel only group, and immunohistochemical staining showed a significant increase in the number of Ki67 positive cells in wound in hUCMSC+gel group compared with those in gel only group (t=4.39, P<0.05), with no statistically significant difference in the number of vimentin positive cells in wound between the 2 groups (P>0.05). Conclusions The application of hyaluronic acid gel containing hUCMSCs to the wound is simple to perform and is therefore a preferable route. Topical application of hUCMSCs can promote healing of the autologous Meek microskin grafted area in patients with extensive burns, shorten wound healing time, and alleviate scar hyperplasia. The above effects may be related to the increased epidermal thickness and epidermal crest, and active cell proliferation.
- Burns /
- Cicatrix /
- Mesenchymal stem cell transplantation /
- Skin transplantation /
- Meek microskin
闫甜甜：实施研究、采集及分析数据、论文撰写；肖荣、王影：研究指导、研究设计、分析数据、论文撰写、经费支持；林国安、郑银、李文军、尚新志、孟进松、胡东升、李嵩、王超、林之琛、陈鸿昌、赵东彦、唐棣：实施研究、采集数据；赵辉：实施研究、文章审阅

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1 大面积烧伤患者2组创面处理。1A.划定包含2组相邻创面（大小均为10 cm×10 cm）的受试区；1B.hUCMSC+凝胶组涂抹含hUCMSC（细胞总数为3×10⁷个）的透明质酸凝胶，单纯凝胶组涂抹单纯透明质酸凝胶；1C.2组创面均移植扩展比例为1∶6的自体Meek微型皮片

注：标记“+”为人脐带间充质干细胞（hUCMSC）+凝胶组，标记“-”为单纯凝胶组

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2 大面积烧伤患者2组创面行Meek微型皮片移植前后愈合情况。2A.术前清创后即刻；2B.术中hUCMSC+凝胶组涂抹含hUCMSC（细胞总数为3×10⁷个）的透明质酸凝胶，单纯凝胶组涂抹单纯透明质酸凝胶，且均移植扩展比例为1∶6的自体Meek微型皮片后；2C、2D、2E.分别为术后2、3、4周，hUCMSC+凝胶组创面愈合面积均较单纯凝胶组大

注：白色虚线上方为人脐带间充质干细胞（hUCMSC）+凝胶组，下方为单纯凝胶组

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3 大面积烧伤患者2组创面行Meek微型皮片移植前后瘢痕情况。3A.术前清创后即刻；3B.术中hUCMSC+凝胶组涂抹含hUCMSC（细胞总数为3×10⁷个）的透明质酸凝胶，单纯凝胶组涂抹单纯透明质酸凝胶，且均移植扩展比例为1∶6的自体Meek微型皮片后；3C、3D.分别为术后3、6个月，与hUCMSC+凝胶组相比，单纯凝胶组瘢痕高度更高，颜色更深；3E.术后12个月，hUCMSC+凝胶组瘢痕颜色、高度均与正常皮肤相近，单纯凝胶组仍可见凸出的淡粉色瘢痕

注：白色虚线上方为人脐带间充质干细胞（hUCMSC）+凝胶组，下方为单纯凝胶组

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4 大面积烧伤患者2组创面行Meek微型皮片移植术后3个月组织学分析。4A. hUCMSC+凝胶组可见表皮嵴，偶见真皮层汗腺、皮脂腺等苏木精-伊红×100；4B.hUCMSC+凝胶组Ki67大量表达于创面基底层、棘层、颗粒层，Ki67阳性细胞数较多辣根过氧化物酶-苏木精×100；4C.hUCMSC+凝胶组真皮层可见大量波形蛋白表达辣根过氧化物酶-苏木精×100；4D.单纯凝胶组表皮层扁平、基底层表皮嵴消失，未见明显皮肤附件苏木精-伊红×100；4E.单纯凝胶组Ki67主要表达于基底层，Ki67阳性细胞数较少辣根过氧化物酶-苏木精×100；4F.单纯凝胶组波形蛋白表达与图4C相近辣根过氧化物酶-苏木精×100

注：人脐带间充质干细胞（hUCMSC）+凝胶组涂抹含hUCMSC（细胞总数为3×10⁷个）的透明质酸凝胶，单纯凝胶组涂抹单纯透明质酸凝胶，随后2组均移植扩展比例为1∶6的自体Meek微型皮片；Ki67和波形蛋白阳性表达均为棕色

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表1 大面积烧伤患者2组创面行Meek微型皮片移植术后各时间点愈合率比较（%，x¯±s）

组别	创面数（个）	2周	3周	4周
hUCMSC+凝胶组	20	80±11	84±12	92±9
单纯凝胶组	20	67±18	74±21	84±16
t值		4.01	3.52	3.66
P值		0.020	0.040	0.040
注：人脐带间充质干细胞（hUCMSC）+凝胶组创面涂抹含有hUCMSC（细胞总数为3×10⁷个）的透明质酸凝胶，单纯凝胶组创面涂抹单纯透明质酸凝胶，且均移植扩展比例为1∶6的自体Meek微型皮片

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表2 大面积烧伤患者2组创面行Meek微型皮片移植术后各时间点创面VSS评分比较（分，x¯±s）

组别	创面数（个）	3个月	6个月	12个月
hUCMSC+凝胶组	20	6.8±1.8	5.6±1.6	4.0±1.4
单纯凝胶组	20	7.8±1.9	6.7±2.1	5.4±1.6
t值		-4.79	-4.37	-5.47
P值		<0.001	<0.001	<0.001
注：VSS为温哥华瘢痕量表；人脐带间充质干细胞（hUCMSC）+凝胶组创面涂抹含hUCMSC（细胞总数为3×10⁷个）的透明质酸凝胶，单纯凝胶组创面涂抹单纯透明质酸凝胶，且均移植扩展比例为1∶6的自体Meek微型皮片

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