The healing of diabetic wounds is poor due to a collagen deposition disorder. Matrix metalloproteinase-9 (MMP-9) is closely related to collagen deposition in the process of tissue repair. Many studies have demonstrated that extracellular vesicles derived from adipose-derived stem cells (ADSC-EVs) promote diabetic wound healing by enhancing collagen deposition.In this study, we explored whether ADSC-EVs could downregulate the expression of MMP-9 in diabetic wounds and promote wound healing by improving collagen deposition. The potential effects of ADSC-EVs on MMP-9 and diabetic wound healing were tested both in vitro and in vivo.We first evaluated the effect of ADSC-EVs on the proliferation and MMP-9 secretion of HaCaT cells treated with advanced glycation end product-bovine serum albumin (AGE-BSA) using CCK-8, western blot and MMP-9 enzyme-linked immunosorbent assay(ELISA). Next, the effects of ADSC-EVs on healing, re-epithelialisation, collagen deposition, and MMP-9 concentration in diabetic wound fluids were evaluated in an immunodeficient mouse model via MMP-9 ELISA and haematoxylin and eosin, Masson's trichrome, and immunofluorescence staining for MMP-9.In vitro, ADSC-EVs promoted the proliferation and MMP-9 secretion of HaCaT cells. In vivo, ADSC-EVs accelerated diabetic wound healing by improving re-epithelialisation and collagen deposition and by inhibiting the expression of MMP-9.ADSC-EVs possess the potential of healing of diabetic wounds in a mouse model by inhibiting downregulating MMP-9 and improving collagen deposition. Thus, ADSC-EVs are a promising candidate for the treatment of diabetic wounds.
In the application of CRISPR genome editing, direct cellular delivery of non-replicable Cas9/sgRNA may reduce unwanted gene targeting and integrational mutagenesis, thus offering greater specificity and safety. Cas9/sgRNA delivery system holds great potential for treating genetic diseases. This review summarizes the advances of Cas9/sgRNA delivery systems and its therapeutic applications, providing new understandings and inspirations for vector design and future clinical applications.
To investigate the effect of adipose-derived stem cell derived exosomes (ADSC-Exos) on angiogenesis after skin flap transplantation in rats.ADSCs were isolated and cultured by enzymatic digestion from voluntary donated adipose tissue of patients undergoing liposuction. The 3rd generation cells were observed under microscopy and identified by flow cytometry and oil red O staining at 14 days after induction of adipogenesis. After cells were identified as ADSCs, ADSC-Exos was extracted by density gradient centrifugation. And the morphology was observed by transmission electron microscopy, the surface marker proteins (CD63, TSG101) were detected by Western blot, and particle size distribution was measured by nanoparticle size tracking analyzer. Twenty male Sprague Dawley rats, weighing 250-300 g, were randomly divided into ADSC-Exos group and PBS group with 10 rats in each group. ADSC-Exos (ADSC-Exos group) and PBS (PBS group) were injected into the proximal, middle, and distal regions of the dorsal free flaps with an area of 9 cm×3 cm along the long axis in the two groups. The survival rate of the flap was measured on the 7th day, and then the flap tissue was harvested. The tissue morphology was observed by HE staining, and mean blood vessel density (MVD) was measured by CD31 immunohistochemical staining.ADSCs were identified by microscopy, flow cytometry, and adipogenic induction culture. ADSC-Exos was a round or elliptical membrane vesicle with clear edge and uniform size. It has high expression of CD63 and TSG101, and its size distribution was 30-200 nm, which was in accordance with the size range of Exos. The distal necrosis of the flaps in the ADSC-Exos group was milder than that in the PBS group. On the 7th day, the survival rate of the flaps in the ADSC-Exos group was 64.2%±11.5%, which was significantly higher than that in the PBS group (31.0%±6.6%; t=7.945, P=0.000); the skin appendages in the middle region of the flap in the ADSC-Exos group were more complete, the edema in the proximal region was lighter and the vasodilation was more extensive. MVD of the ADSC-Exos group was (103.3±27.0) /field, which was significantly higher than that of the PBS group [(45.3±16.2)/field; t=3.190, P=0.011].ADSC-Exos can improve the blood supply of skin flaps by promoting the formation of neovascularization after skin flap transplantation, thereby improve the survival rate of skin flaps in rats.探讨脂肪干细胞来源外泌体（adipose-derived stem cell derived exosomes，ADSC-Exos）对大鼠皮瓣移植后血管新生的影响。.取抽脂术患者自愿捐赠脂肪组织，采用酶消化法分离培养 ADSCs；取第 3 代细胞光镜观察形态，流式细胞术鉴定细胞表面标志物，成脂诱导培养 14 d 后油红 O 染色观察。细胞鉴定为 ADSCs 后，采用密度梯度离心法提取 ADSC-Exos；透射电镜观察形态，Western blot 检测膜表面标志蛋白（CD63、TSG101），纳米颗粒跟踪分析仪测量其粒径分布。取 20 只雄性 SD 大鼠，体质量 250～300 g，随机分为 ADSC-Exos 组和 PBS 组，每组 10 只。两组大鼠背部沿长轴方向制作面积为 9 cm×3 cm 随意皮瓣后，分别于近端、中间和远端区域注射 ADSC-Exos（ADSC-Exos 组）或 PBS（PBS 组）。术后大体观察皮瓣成活情况，第 7 天测算皮瓣成活率后切取皮瓣组织，HE 染色观察组织形态，CD31 免疫组织化学染色测定皮瓣微血管密度（mean blood vessel density，MVD）。.光镜、流式细胞术及成脂诱导培养鉴定培养细胞为 ADSCs。ADSC-Exos 为边缘清晰、大小形态均匀的圆形或椭圆形膜性囊泡，高表达标志蛋白 CD63 和 TSG101，粒径分布在 30～200 nm，符合外泌体粒径范围。术后两组皮瓣远端均出现不同程度坏死表现，但 ADSC-Exos 组程度较 PBS 组轻；第 7 天 ADSC-Exos 组皮瓣成活率为 64.2%±11.5%，显著大于 PBS 组的 31.0%±6.6%（t=7.945，P=0.000）；中间区域皮肤附属器官更完整，近端区域组织水肿程度轻、血管扩张更多。ADSC-Exos 组 MVD 为（103.3±27.0）个/视野，显著多于 PBS 组（45.3±16.2）个/视野（t=3.190，P=0.011）。.ADSC-Exos 可通过促进皮瓣移植后新生血管的形成，改善皮瓣血供，进而提高大鼠皮瓣成活率。.
To investigate the effect of circulating estrogen level on the outcome of free fat grafting in nude mice.Eighteen female nude mice aged 6-8 weeks (weighing, 20-25 g) were randomly divided into 3 groups ( n=6). The nude mice in the ovariectomized group were treated with ovariectomy. The nude mice in the high estrogen group and the normal estrogen group only made the same incision to enter the peritoneum without ovariectomy. The nude mice in the high estrogen group were given the estradiol (0.2 mg/g) every 3 days for 30 days. The other two groups were given the same amount of PBS every 3 days. At 30 days after operation, the tail vein blood of nude mice in 3 groups were detected by estradiol ELISA kit, and the free fat (0.3 mL) donated by the females was injected into the sub-scalp of nude mice. After 8 weeks of fat grafting, the samples were taken for gross observation and weighing, and the prepared slices were stained with HE staining, CD31-perilipin fluorescence staining, immunohistochemical staining of uncoupling protein 1 (UCP1), and immunofluorescence staining of estrogen receptor α. The diameter of adipocytes and vascular density of adipose tissue were measured. The mRNA expressions of UCP1 and estrogen receptor α were detected by realtime fluorescence quantitative PCR (qRT-PCR).All nude mice survived during experiment. ELISA test showed that the concentration of estradiol significantly decreased in the ovariectomized group and increased in the high estrogen group compared with the normal estrogen group ( P<0.05). At 8 weeks after fat grafting, the graft volume from large to small was ovariectomized group, normal estrogen group, and high estrogen group. There was significant difference in wet weight between the ovariectomized group and high estrogen group ( P<0.05). Section staining showed that compared with the normal estrogen group, the adipocytes in the ovariectomized group were larger, the expression of peri-lipoprotein was weaker, the vascular density decreased, and the expressions of UCP1 was negative, and the estrogen receptor α positive cells reduced. The above observation results in the high estrogen group were contrary to those in the ovariectomized group. There were significant differences in the diameter of adipocytes, the vascular density of adipose tissue, the number of the estrogen receptor α positive cells between groups ( P<0.05). The results of qRT-PCR showed that the mRNA expressions of UCP1 and estrogen receptor α significantly increased in the high estrogen group and decreased in the ovariectomized group compared with the normal estrogen group, and the differences were significant ( P<0.05).The level of circulating estrogen has a significant effect on the outcome of free fat grafting in nude mice. Low estrogen level leads to hypertrophy of graft adipocytes, while high estrogen level leads to the production of a large amount of beige fat and high vascular density in fat grafts, which may be related to the activation of estrogen receptor α on adipocytes.探讨循环雌激素水平对裸鼠颗粒脂肪移植转归的影响。.取 6～8 周龄雌性裸鼠 18 只（体质量 20～25 g），随机分为 3 组（ n=6）。去势组采用卵巢切除术制备去势裸鼠模型，高雌激素组及正常雌激素组仅作相同切口进入腹膜后，不切除卵巢组织。术后高雌激素组每 3 天灌胃雌二醇（0.2 mg/g）1 次，共持续 30 d；其他两组同时间点等量 PBS 灌胃。术后 30 d 取 3 组裸鼠尾静脉血经 ELISA 检测雌二醇浓度后，将接受抽脂术的健康女性捐赠的颗粒脂肪注入裸鼠头皮下（0.3 mL/只）。脂肪移植后 8 周取材大体观察、称重后，制备切片行 HE 染色、CD31-围脂滴蛋白荧光染色、解耦联蛋白 1（uncoupling protein 1，UCP1）免疫组织化学染色和雌激素受体 α 免疫荧光染色，测量脂肪细胞直径及脂肪组织血管密度；实时荧光定量 PCR 检测 UCP1 和雌激素受体 α mRNA 表达。.实验期间裸鼠无死亡。ELISA 检测显示与正常雌激素组相比，去势组雌二醇浓度明显降低，高雌激素组明显升高，差异均有统计学意义（ P<0.05）。脂肪移植 8 周后，移植物体积从大到小依次为去势组、正常雌激素组、高雌激素组，其中去势组移植物湿重显著高于高雌激素组（ P<0.05）。组织学染色显示，与正常雌激素组相比，去势组脂肪细胞增大且围脂滴蛋白表达较弱、血管密度减小，几乎不表达脂肪标记物 UCP1，雌激素受体 α 阳性细胞减少；而高雌激素组上述观察结果与去势组相反；脂肪细胞直径、血管密度及雌激素受体 α 阳性细胞组间比较，差异均有统计学意义（ P<0.05）。实时荧光定量 PCR 检测显示，与正常雌激素组相比，高雌激素组移植物 UCP1 和雌激素受体 α mRNA 相对表达量升高，而去势组均降低；组间比较差异均有统计学意义（ P<0.05）。.体内循环雌激素水平对脂肪移植的转归有显著影响。低雌激素水平导致移植物脂肪细胞肥大，高雌激素水平导致脂肪移植物中生成大量米色脂肪并伴随血管密度增大，其机制可能与升高的循环雌激素水平活化脂肪细胞上雌激素受体 α 相关。.
Abstract Background In order to establish the link between quality traits and genotypes of Blumea balsamifera and specific allele variations, 51 B. balsamifera germplasm resources were used to evaluate the quality traits, EST-SSR markers were used to analyze the genetic diversity, population structure and the correlation between main quality traits and EST-SSR molecular markers. Results (1) There were abundant variations in the main quality traits of the tested materials. The highest coefficient of variation was Eriodictyol (85.5%), followed by carotenoid 3,3’,5,7-tetrahydroxy-4’-methoxyflavanone and Sakuranetin, 11 excellent B. balsamifera germplasms were selected by principal component analysis; (2) Genetic diversity analysis showed that a total of 102 alleles were amplified from 22 pairs of primers, of which the effective alleles accounted for 53.52%, and the average polymorphism information content was 0.488, 9 pairs of primers had high polymorphism (PIC > 0.5), 11 pairs of primers had moderate polymorphism (0.25 < PIC < 0.5), and the proposed primers had strong effectiveness and good polymorphism; The average gene flow Nm was 0.203, which was far less than 1, indicating that there was almost no inbreeding between germplasms; The average Nei diversity index and Shannon information index were 0.542 and 1.023, which showed that the population had a high level of genetic diversity; (3) UPGMA cluster analysis and population structure analysis divided the 51 germplasms into 4 groups, and the germplasms from the same source were often gathered in a group. UPGMA cluster analysis showed that geographic distance affected the genetic relationship of germplasm to some extent; PCA analysis indicated that the genetic background of germplasm with similar geographical distance was similar, and the genetic relationship was closer; The analysis of population structure showed that the geographical origin of germplasm was closely related to the genetic relationship of germplasm, and again confirmed the accuracy of UPGMA cluster analysis; (4) The result of linkage disequilibrium(LD) analysis showed that the markers with D '>0.5 accounted for more than 50%, and the recombination probability between germplasm genes was low, indicating that the level of genetic diversity of the population was high, suggesting that the experimental materials were suitable for association analysis, and (5) The result of correlation analysis between quality traits and EST-SSR markers showed that 23 markers significantly correlated with 6 quality traits were detected, and the variance interpretation rate was 19.33% − 57.86%. Among them, the character Blumeatin had the best correlation with EST-SSR loci, and showed a very significant correlation with Bbf377 marker primer. Conclusion The results could lay a theoretical foundation for the selection and genetic improvement of excellent germplasm of B. balsamifera in the future.
We applied mRNA Differential Display to screen different genes expression during early embryonic stage of chicken and quail hybridization,we cloned the differential gene segments and analysed these sequences' relationship with the embryonic development of the early stage hybridization,and presumed the main infection to the death of the early embryo.The result shows that six genes have high homology with several chicken mRNA.One gene segment of 364bp contrasted with Gene bank,observed that it is highly conformability with estrogen receptor binding site associated antigen mRNA.And the other 229bp gene is similar to peptidyl prolyl isomerase enzyme mRNA.We found the estrogen receptor binding site associated antigen mRNA may have main infection with the early embryonic development.
Background: Growing evidence has demonstrated that adipose-derived stem cell-derived extracellular vesicles enhance the survival of fat grafts and the browning of white adipose tissue. We evaluated whether supplementation with adipose-derived stem cell-derived extracellular vesicles promotes the survival and browning of fat grafts. Methods: Extracellular vesicles derived from adipose-derived stem cells were injected into fat grafts of C57BL/6 mice once per week until postgraft week 12. The grafts were collected and weighed after postgraft weeks 2, 4, and 12. The histological morphology, neovascularization, and the proportion of M2 macrophages of grafts were evaluated. The ability of extracellular vesicles to promote macrophage polarization and catecholamine secretion was detected. Whether the inducement of browning adipose differentiation is extracellular vesicles or the paracrine effect of M2 macrophages polarized by extracellular vesicles was also verified. Results: Grafts treated by extracellular vesicles derived from adipose-derived stem cells showed enhanced beige adipose regeneration with increased neovascularization, M2 macrophage proportion, and norepinephrine secretion at postgraft week 4. Increased retention and decreased fibrosis and necrosis were noted at postgraft week 12. The extracellular vesicles uptake by macrophages promoted M2 type polarization and catecholamine secretion while suppressing M1 type polarization. Of note, browning adipose differentiation with enhanced energy expenditure could be promoted only by the conditioned medium from extracellular vesicle–polarized M2 macrophages but not by extracellular vesicles themselves. Conclusions: Supplementation with extracellular vesicles derived from adipose-derived stem cells increases fat graft survival and browning by which extracellular vesicles–polarized M2 macrophages secrete catecholamines to promote beige adipose regeneration.
The genus Blumea is one of the most economically important genera of Inuleae (Asteraceae) in China. It is particularly diverse in South China, where 30 species are found, more than half of which are used as herbal medicines or in the chemical industry. However, little is known regarding the phylogenetic relationships and molecular evolution of this genus in China. We used nuclear ribosomal DNA (nrDNA) internal transcribed spacer (ITS) and chloroplast DNA (cpDNA) trnL-F sequences to reconstruct the phylogenetic relationship and estimate the divergence time of Blumea in China. The results indicated that the genus Blumea is monophyletic and it could be divided into two clades that differ with respect to the habitat, morphology, chromosome type, and chemical composition of their members. The divergence time of Blumea was estimated based on the two root times of Asteraceae. The results indicated that the root age of Asteraceae of 76-66 Ma may maintain relatively accurate divergence time estimation for Blumea, and Blumea might had diverged around 49.00-18.43 Ma. This common ancestor had an explosive expansion during the Oligocene and Miocene and two major clades were differentiated during these epochs 29.60 Ma (17.76-45.23 Ma 95% HPD (Highest Posterior Density). Evidence from paleogeography and paleoclimate studies has confirmed that Blumea experienced differentiation and an explosive expansion during the Oligocene and Miocene.
Objective:To determine the suitable testing methods of germination and viability of Glehnia littoralis Fr.Schmidt ex Miq.seed.Methods:The appropriate seed germination conditions were obtained by testing the influence of the different germination beds and temperatures on the average germination rate.The appropriate testing method of seed viability was found out by observing the effects of different concentration and temperature of TCC on the seed dye rate.Results:The appropriate conditions for seed germination are at 25℃on filter paper.The best testing conditions of seed viability are dying for 5 hours in0.6%TCC at 30℃.Conclusion:Based on the research of germination conditions and testing method of seed viability,our study offers help for the establishment of testing rules and quality standard of G.littoralis seed.
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