人脐带间充质干细胞微囊减轻小鼠急性肾损伤的研究

doi:10.3877/cma.j.issn.2095-1221.2018.05.002

目的

探讨人脐带间充质干细胞（MSCs）源性细胞外囊泡Oct-4 mRNA对受损的肾小管上皮细胞修复的作用及相关机制。

方法

将培养的缺氧损伤肾小管上皮细胞置于含有人脐带MSCs细胞外囊泡及不同对照培养液的培养腔室玻片上孵育48?h，应用BrdU及TUNEL染色，检测各组细胞增殖或凋亡情况。将急性肾损伤模型小鼠分为4组：空白组、EVs组、Oct-4过表达组、Oct-4低敲组。并按照分组分别注射磷酸盐缓冲液（Vehicle），人脐带MSCs细胞外囊泡（EVs），过表达Oct-4基因的人脐带MSCs细胞外囊泡（EVs?+?Oct-4）及敲除Oct-4基因的人脐带MSCs外囊泡（EVs-Oct-4），并在注射48?h及2周后采血测肌酐（Crea）及尿素氮（BUN），了解肾功能变化；对各组上述处理后的肾组织应用TUNEL与增殖细胞核抗原表达量检测各组肾脏细胞凋亡与增殖情况；通过Masson染色检测了各组肾脏纤维化程度；通过PCR探索肾损伤后肾组织细胞Snail基因的表达变化。数据分析采用方差分析和SNK-q检验。

结果

EVs?+ Oct-4处理缺氧的肾小管上皮细胞48?h后，TUNEL染色显示具有最少的凋亡细胞数（0~1）/?HPF，BrdU显示有最多的增殖细胞（7±2）/HPF。EVs，EV-Oct-4以及Vehicle对体外缺氧肾小管上皮细胞的上述作用依次减弱（P?<?0.01）。急性肾损伤后，注射EVs?+?Oct-4可降低肾损伤后Crea水平（28?mmol/L）和BUN水平（17?mmol/L）；并抑制肾组织纤维化，使Masson染色阳性面积减少至（3.2±1.0）﹪；同时提高肾组织PCNA染色阳性细胞数（损伤48h后为（70±7）/HPF，2周后为（21±4）/HPF，减少TUNEL染色阳性细胞数[损伤48?h后为（2±1）/HPF，2周后为（3±2）/?HPF]。注射EVs，EV-Oct-4以及Vehicle对产生上述效应的作用依次减弱（P?<?0.01）。PCR结果表明，Vehicle组肾小管上皮细胞Snail表达最高2.3±0.2，而EVs + Oct-4则可降低Snail表达0.7±0.1。

结论

EVs可通过抑制凋亡促进增殖、抑制纤维化治疗肾损伤，而这其中EVs包含的OCT-4 mRNA可通过抑制肾损伤后Snail基因的表达来抑制肾脏纤维化的形成。

关键词: 急性肾损伤, 间充质干细胞, 细胞外囊泡

Objectives

To explore the effect of Oct-4 overexpressing mesenchymal stem cell derived extracellular vesicles (MSC-EVs) on acute kidney injury (AKI) and the related mechanism.

Methods

The renal tubular epithelial cells treated in low oxygen environment were cocultured with MSC-EVs and control medium for 48 hours. BrdU and TUNEL were used to assess cell proliferation and apoptosis. Mice with acute kidney injury were randomly divided into 4 groups, which were injected with "Vehicle", "EVs", Oct-4 overexpressing EVs (EVs + Oct-4) , and Oct-4 knocked-down EVs (EVs–Oct-4) . Blood creatinine and urine nitrone levels were assessed 48 hours after injection. The mice kedneys were harvested for TUNEL and PCNA staining to evaluate apoptosis and proliferation. Masson trichrome staining was used to evaluate renal fibrosis. Snail gene expression was assessed by PCR. The data was analyzed by ANOVA test and SNK-q test.

Results

48 hours after hypoxic treatment, TUNEL showed the EVs + Oct-4 group has a lowest apoptosis level (0-1) /?HPF. BrdU showed the EVs?+?Oct-4 group has a highest proliferation level (7±2) /HPF. Injection of Oct-4 overexpressing EVs could significantly decrease the level of blood Crea (28?mmol/?L) and BUN (17?mmol/L) , and the kidney fibrosis was also alleviated, as shown by slight Masson staining (3.2±1.0) ﹪ and few PCNA positive cells (70±7) /HPF 48?h post AKI, (21±4) /HPF 2?week post AKI) , the number of TUNEL positive cells was decreased (2±1) /HPF 48?h post AKI, (3±2) /?HPF 2?week post AKI. PCR showed that Vehicle group had a highest Snail level 2.3±0.2, and the EVs?+ Oct-4 group had a lowest Snail level 0.7±0.1.

Conclusions

MSC-EVs provides therapeutic effects in ischemic-reperfusion injury. Oct-4 overexpression could enhance its therapeutic effects by inhibiting apoptosis, promoting proliferation, and inhibiting fibrosis.

Key words: Acute kidney injury, Mesenchymal stem cells, Extracellular vesicles

图1 荧光显微镜下观察缺氧处理后肾小管上皮细胞TUNEL和BrdU染色（×200）

表1 各组肾损伤48?h后TUNEL和BrdU染色情况比较（±s，/HPF）

分组	样数	TUNEL染色	BrdU染色
空白组	6	10±2	0 ~ 1
EVs组	6	2±1^a	4±1^a
Oct-4过表达组	6	0 ~1^ab	7±2^ab
Oct-4低敲组	6	7±2^abc	2±1^abc
F值	?	50.20	30.57
P值	?	< 0.01	< 0.01

表1 各组肾损伤48?h后TUNEL和BrdU染色情况比较（±s，/HPF）

分组	样数	TUNEL染色	BrdU染色
空白组	6	10±2	0 ~ 1
EVs组	6	2±1^a	4±1^a
Oct-4过表达组	6	0 ~1^ab	7±2^ab
Oct-4低敲组	6	7±2^abc	2±1^abc
F值	?	50.20	30.57
P值	?	< 0.01	< 0.01

图2 光学显微镜下观察缺氧再灌注后各组小鼠的细胞凋亡情况（TUNEL染色，×200）

表2 各组肾损伤48 h及2周后TUNEL和BrdU染色情况比较（±s，/HPF）

分组	样数	TUNEL染色		PCNA染色
分组	样数	48 h	2周	48 h	2周
空白组	6	224±25	145±20	5± 2	2±1
EVs组	6	114±23^a	88±12^a	51± 6^a	10±2^a
Oct-4过表达组	6	2± 1^ab	3± 2^ab	70± 7^ab	21±4^ab
Oct-4低敲组	6	134±12^ac	107±14^abc	29±10^abc	5±2^bc
F值	?	153.74	116.29	99.93	66.88
P值	?	< 0.01	< 0.01	< 0.01	< 0.01

表2 各组肾损伤48 h及2周后TUNEL和BrdU染色情况比较（±s，/HPF）

分组	样数	TUNEL染色		PCNA染色
分组	样数	48 h	2周	48 h	2周
空白组	6	224±25	145±20	5± 2	2±1
EVs组	6	114±23^a	88±12^a	51± 6^a	10±2^a
Oct-4过表达组	6	2± 1^ab	3± 2^ab	70± 7^ab	21±4^ab
Oct-4低敲组	6	134±12^ac	107±14^abc	29±10^abc	5±2^bc
F值	?	153.74	116.29	99.93	66.88
P值	?	< 0.01	< 0.01	< 0.01	< 0.01

图3 光学显微镜下观察急性肾损伤后各组细胞的增殖情况（PCNA染色，×200）

图4 急性肾损伤48 h及2周后小鼠血尿素氮及肌酐含量

图5 光学显微镜下观察急性肾损伤后各组肾脏纤维化情况（×200）

图6 RT-qPCR检测急性肾损伤后肾组织中Snail基因表达结果

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Extracellular vesicles derived from human umbilical cord mesenchymal stem cells ameliorate acute kidney injury in mice

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Extracellular vesicles derived from human umbilical cord mesenchymal stem cells ameliorate acute kidney injury in mice