Objective
To determine the efficacy of D980-nm laser in dissolving fat and renewing skin, and to explore the clinical application of D980-nm laser in reconstruction of photodamaged skin.
Methods
Eighteen 12-14 month-old male Sprague-Dawley rats, weighing 400-450 g, were randomly divided into 3 groups (n=6). The rat skin at the left side was exposed to D980-nm laser irradiation at a density of 20 J/cm2, a power of 8 W, a pulse width of 20 ms, and a pulse frequency of 40 Hz for 1 time (group A), 2 times of 5-minute interval (group B), and 3 times of 5-minute interval (group C) as a treatment course, for 4 treatment courses with an interval of 1 week; the other side of the skin was not treated as the control groups (groups A1, B1, and C1, respectively). After 8 weeks, the skin was harvested for HE staining and immunohistochemical staining to observe the structure changes of skin, to measure the dermal thickness, to count the number of fibroblasts, and detect the expressions of transforming growth factor β1 (TGF-β1) and basic fibroblast growth factor (bFGF).
Results
Compared with groups A1, B1, and C1, the skin structure was significantly improved in groups A, B, and C. After D980-nm laser irradiation, the number of fat cells decreased; local angiogenesis was observed; the total number of fibroblasts and fibers increased; the collagen fiber had large diameter, and arranged closely and regularly; the dermal thickness and the number of the fibroblasts increased; and the expressions of TGF-β1 and bFGF were significantly enhanced, showing significant differences (P<0.05). With increased D980-nm laser irradiation times, the above indexes increased, showing significant differences between group C and groups A, B (P<0.05).
Conclusion
D980-nm laser treatment has lipolytic and tender effect on the skin, and the frequency of the treatment is an important factor in skin renewal.
Objective To investigate whether the Runx2 gene can induce the differentiation of human amniotic mesenchymal stem cells (hAMSCs) to ligament fibroblasts in vitro and promote the tendon-bone healing in rabbits. Methods hAMSCs were isolated from the placentas voluntarily donated from healthy parturients and passaged, and then identified by flow cytometric identification. Adenoviral vectors carrying Runx2 gene (Ad-Runx2) and empty vector adenovirus (Ad-NC) were constructed and viral titer assay; then, the 3rd generation hAMSCs were transfected with Ad-Runx2 (Ad-Runx2 group) or Ad-NC (Ad-NC group). The real-time fluorescence quantitative PCR and Western blot were used to detect Runx2 gene and protein expression to verify the effectiveness of Ad-Runx2 transfection of hAMSCs; and at 3 and 7 days after transfection, real-time fluorescence quantitative PCR was further used to detect the expressions of ligament fibroblast-related genes [vascular endothelial growth factor (VEGF), collagen type Ⅰ, Fibronectin, and Tenascin-C]. The hAMSCs were used as a blank control group. The hAMSCs, hAMSCs transfected with Ad-NC, and hAMSCs were mixed with Matrigel according to the ratio of 1 : 1 and 1 : 2 to construct the cell-scaffold compound. Cell proliferation was detected by cell counting kit 8 (CCK-8) assay, and the corresponding cell-scaffold compound with better proliferation were taken for subsequent animal experiments. Twelve New Zealand white rabbits were randomly divided into 4 groups of sham operation group (Sham group), anterior cruciate ligament reconstruction group (ACLR group), anterior cruciate ligament reconstruction+hAMSCs transfected with Ad-NC-scaffold compound group (Ad-NC group), and anterior cruciate ligament reconstruction+hAMSCs transfected with Ad-Runx2-scaffold compound group (Ad-Runx2 group), with 3 rabbits in each group. After preparing the ACL reconstruction model, the Ad-NC group and the Ad-Runx2 group injected the optimal hAMSCs-Matrigel compunds into the bone channel correspondingly. The samples were taken for gross, histological (HE staining and sirius red staining), and immunofluorescence staining observation at 1 month after operation to evaluate the inflammatory cell infiltration as well as collagen and Tenascin-C content in the ligament tissues. ResultsFlow cytometric identification of the isolated cells conformed to the phenotypic characteristics of MSCs. The Runx2 gene was successfully transfected into hAMSCs. Compared with the Ad-NC group, the relative expressions of VEGF and collagen type Ⅰ genes in the Ad-Runx2 group significantly increased at 3 and 7 days after transfection (P<0.05), Fibronectin significantly increased at 3 days (P<0.05), and Tenascin-C significantly increased at 3 days and decreased at 7 days (P<0.05). CCK-8 detection showed that there was no significant difference (P>0.05) in the cell proliferation between groups and between different time points after mixed culture of two ratios. So the cell-scaffold compound constructed in the ratio of 1∶1 was selected for subsequent experiments. Animal experiments showed that at 1 month after operation, the continuity of the grafted tendon was complete in all groups; HE staining showed that the tissue repair in the Ad-Runx2 group was better and there were fewer inflammatory cells when compared with the ACLR group and the Ad-NC group; sirius red staining and immunofluorescence staining showed that the Ad-Runx2 group had more collagen typeⅠ and Ⅲ fibers, tending to form a normal ACL structure. However, the fluorescence intensity of Tenascin-C protein was weakening when compared to the ACLR and Ad-NC groups. Conclusion Runx2 gene transfection of hAMSCs induces directed differentiation to ligament fibroblasts and promotes tendon-bone healing in reconstructed anterior cruciate ligament in rabbits.
In order to investigate the effects of mechanical stretching combined with prostaglandin E2 (PGE2) on the gene expression of lysyl oxidases (LOXs) in keratoconus, we treated cultured corneal fibroblasts from healthy human cornea and keratoconus patient cornea with PGE2 and/or cyclic stretch (12% elongation, 0.1 Hz, 12 h). Real-time fluorescent quantitative polymerase chain reaction was used to detect the gene expression of LOXs. The results showed that the gene expression of LOXs in keratoconus group was significantly lower than that in the healthy one. Compared to the static control group, 12% stretching alone up-regulated gene expression of LOXL-2, LOXL-4 in the healthy group, whereas it down-regulated LOXL-3, LOXL-4 in the keratoconus group. Combination of 12% stretching and PEG2 induced LOXL-4 down-regulation in in healthy group, and all LOXs except LOXL-1 in keratoconus group. The results suggested that combination of mechanical stretching and PGE2 down-regulate the gene expression of LOXs in keratoconus. Lower LOXs expression may lead to impaired cross-linking, and thus to a loss of cohesion between collagen fibrils, affecting corneal structural stability by collagen lamellae slippage. This may facilitate the development of keratoconus. Exploring the effects of mechanical stretching and inflammatory factor on the expression LOXs in this paper will help us to understand the possible mechanism of how the keratoconus occurs and develops well, and provide the reference for the prevention and treatment of keratoconus.
ObjectiveTo investigate the effect of microRNA-135a (miR-135a) in human amnion mesenchymal stem cell exosome (hAMSC-Exo) on the migration of fibroblasts.MethodsThe hAMSC-Exo was extracted with exosomes separation kit and identified, the effect of hAMSC-Exo on fibroblasts migration was detected by scratch test. Real-time fluorescence quantitative PCR (qRT-PCR) was used to detect the relative expression of miR-135a gene in hAMSC-Exo after overexpression of miR-135a. Scratch test was used to detect the effect of hAMSC-Exo on the migration of fibroblasts after overexpression and knockdown of miR-135a. Western blot was used to detect the migration related proteins of fibroblasts [large tumor suppressor 2 (LATS2), E-cadherin, N-cadherin, and α smooth muscle actin (α-SMA)] after overexpression and knockdown of miR-135a. The 293T cell exosomes and hAMSC-Exo were used as control.ResultshAMSC-Exos were extracted successfully. Scratch test results showed that hAMSC group had the strongest ability to promote fibroblasts migration, and GW4869 (exosome inhibitor) treatment group had reduced ability to promote fibroblasts migration. qRT-PCR test showed that the relative expression of miR-135a gene in hAMSC-Exo increased significantly after over expression of miR-135a. Scratch test results showed that after over expression of miR-135a, hAMSC-Exo enhanced the migration ability of fibroblasts, while after knockdown of miR-135a, hAMSC-Exo weakened the migration ability of fibroblasts. Western blot results showed that the expressions of E-cadherin, N-cadherin, LATS2 were down regulated and α-SMA was up regulated in each hAMSC-Exo treatment group when compared with 293T cell exosomes group; after over expression of miR-135a, hAMSC-Exo decreased the expressions of E-cadherin, N-cadherin, LATS2 and increased the expression of α-SMA; while after knockdown of miR-135a, the ability of hAMSC-Exo was weakened.ConclusionmiR-135a in hAMSC-Exo can promote fibroblasts’ migration, inhibit the expressions of E-cadherin, N-cadherin, LATS2, and promote the expression of α-SMA.
ObjectiveTo investigate the effect of Smad4 on the fibrosis of tendon derived fibroblasts (TDFs) induced by transforming growth factor β1(TGF-β1) by targeted regulation of miRNA219-5P (miR219-5P).
MethodsThe tendons donated by the volunteers were harvested to isolate and culture TDFs. The 3rd generation cells were used for experiment. Chemically synthesized miR219-5P mimics, miR219-5P inhibitor, and negative control sequences were transfected into TDFs. The gene expression of miR219-5P in TDFs was detected by real-time PCR, and the protein expression of Smad4 in TDFs was detected by Western blot at 48 hours after transfection. The combining sites of miR219-5P and Smad4 in 3'UTR district were predicted by informatics software. Wild type and mutant type reporter gene expression vectors were constructed and then targeted verification was carried out by the luciferase reporter gene test. Transfected TDFs were then induced by TGF-β1. The proliferation activity of the cells were measured by the cell counting kit 8 after culturing for 24, 48, and 72 hours. The expressions of fibrosis related proteins in TDFs were detected by Western blot at 72 hours.
ResultsAfter TDFs were transfected by miR219-5P mimics, miR219-5P expression was significantly up-regulated, but the expressions of Smad4 was decreased subsequently (P<0.05). Intracellular expression of miR219-5P was inhibited by miR219-5P mimics inhibitor, however, the protein expression of Smad4 was significantly increased (P<0.05). Luciferase reporter gene test showed that luciferase activities were significantly decreased in pGL3-WT-Smad4+mimics group, but were significantly increased in pGL3-WT-Smad4+inhibitor group when compared with pGL3-WT-Smad4 transfected group (P<0.05), but no significant difference was found between GL3-MT-Smad4+mimics and pGL3-MT-Smad4+inhibitor groups (P>0.05). Cell proliferation and the fibrosis related proteins were increased in TGF-β1 induced TDFs, however, decreased in TGF-β1 induced TDFs after transfected by miR219-5P inhibitor (P<0.01).
ConclusionmiR219-5P can significantly inhibit fibrosis of TDFs induced by TGF-β1 by down-regulating Smad4 expression.
Objective
To establish a safe, effective, and economic feeder-free culture system which is suitable for the culture of human parthenogenetic embryonic stem cells (hPESCs) in vitro.
Methods
hPESCs were cultured with mTeSRTMl medium (control group) and human foreskin fibroblasts-conditional medium (hFFs-CM) (experimental group). The growth status of hPESCs in both feeder-free culture systems were observed with inverted microscope. Alkaline phosphatase (ALP) analysis and karyotype analysis were used to study the biological characteristics of hPESCs. The expression of hPESCs pluripotent marker Oct-4 was analyzed by RT-PCR. Differentiation experiment in vivo and in vitro was applied to observe the differentiation potential of hPESCs into three germ layers.
Results
hPESCs had regular morphology with difficulty in differentiation in both culture systems. No obvious difference was observed in morphology and expansion speed of hPESCs between 2 groups. After subcultured for 15 passages in vitro, hPESCs in 2 groups could maintain normal female diploid karyotype 46, XX and pluripotency. The expression of Oct-4 mRNA was positive in 2 groups. hPESCs in 2 groups could form embryonic body in differentiation experiment in vitro and could develop into teratomas containing three germ layers in nude mice.
Conclusion
Feeder-free culture system of hFFs-CM can sustain the growth of hPESCs and keep hPESCs undifferentiated state for long. A feeder-free culture system of hPESCs is successfully established, which can support the growth of hPESCs, reduce the contamination from animals, decrease the cost of culture, and satisfy the clinical large-scale application.
Objective To investigate the effects of NGF on the prol iferation, mitotic cycle, collagen synthesis and migration of human dermal fibroblasts (HDFs), and to explore the function of NGF on the wound heal ing. Methods The 3rd generation of HDFs were incubated with various concentrations of NGF (0, 25, 50, 100, 200 and 400 ng/mL), the cell prol iferation was measured with MTT assay. After treated with NGF at 0, 100 ng/mL, the cell cycle of HDFs was determined by flow cytometry (FCM). Hydroxyprol ine and real-time fluorescence quantitative PCR (FQ-PCR) were used to measure collagen synthesis at protein level and mRNA level respectively. The in vitro cell scratch wound model was set up to observe the effect of NGF (0, 50, 100 and 200 ng/mL) on the migration of HDFs after 24 hours of culture. Results Absorbance value of HDFs for different concentrations of NGF (0, 25, 50, 100, 200, and 400 ng/ mL) showed that NGF did not influence the prol iferation of HDFs (P gt; 0.05). When HDFs were treated with NGF at 0 and 100 ng/mL, the result of FCM analysis showed that percentage of HDFs in G0/G1, S, G2/M phases were not changed (P gt; 0.05). Compared with control group, the expression of Col I and Col III were not significantly different, measured by both hydroxyprol ine and FQ-PCR (P gt; 0.05). The rates of HDFs’ migration at various concentrations of NGF (0, 50, 100, 200 ng/ mL) were 52.12% ± 6.50%, 80.67% ± 8.51%, 66.33% ± 3.58%, and 61.19% ± 0.97%, respectively, indicating that NGF could significantly enhanced the migration of HDFs at 50 and 100 ng/mL (P lt; 0.05). Conclusion NGF does not influence prol iferation, mitotic cycle and collagen synthesis of HDFs, but significantly enhanced migration in an in vitro model of wounded fibroblasts.
ObjectiveTo optimize the culture method of human primary pancreatic ductal adenocarcinoma (PDAC) cells and cancer associated fibroblasts (CAFs) and investigate the effect of CAFs on the growth of primary PDAC cells in vitro and tumor formation in patient-derived xenograft (PDX) model.MethodsThe PDAC specimens were collected and primarily cultured. In order to observe the effect of CAFs on the growth of primary PDAC cells in vitro, the CAFs were co-cultured with primary PDAC cells consistently and the alone cultured primary PDAC cells served as the control. Then, these cells were injected into the shoulder blades of NOG mice in order to develop the PDX model.ResultsWhen the primary PDAC cells separated from the CAFs, the proliferation capacity of the primary PDAC decreased rapidly in the passage culture in vitro, and the most cells were terminated within 5 generations. By contrast, when the CAFs co-cultured with the primary PDAC cells, the proliferation capacity of primary PDAC cells were preserved, which could be stably transferred to at least 10 generations. The tumors of NOG mice were detected during 2–3 weeks after injecting the mixed cells (primary PDAC plus CAFs), while had no tumor formation after injecting CAFs alone. The rate of tumor was 92.9% (13 cases) in the primary PDAC plus CAFs group, which was higher than that of the CAFs alone group (64.3%, 9 cases), but there was no statistical difference because of the small sample size. The volume of tumor in the primary PDAC plus CAFs group at 2, 4, 6, and 8 weeks after the tumor cells injection was significantly larger than that in the CAFs alone group at the corresponding time point, the differences were statistically significant (P<0.01).ConclusionsThe CAFs could promote the growth of primary PDAC cells in vitro. This new method of co-culture CAFs with primary PDAC could improve the success rate of primary PDAC cells culture and improve the success rate of PDX model in NOG mice.
Objective To investigate whether human amniotic mesenchymal stem cells (hAMSCs) have the characteristics of mesenchymal stem cells (MSCs) and the differentiation capacity into ligament fibroblastsin vitro. Methods The hAMSCs were separated through trypsin and collagenase digestion from placenta, the phenotypic characteristics of hAMSCs were detected by flow cytometry, the cytokeratin-19 (CK-19) and vimentin expression of hAMSCs were tested through immunofluorescence staining. The hAMSCs at the 3rd passage were cultured with L-DMEM/F12 medium containing transforming growth factor β1 (TGF-β1) and vascular endothelial growth factor (VEGF) as the experimental group and with single L-DMEM/F12 medium as the control group. The morphology of hAMSCs was observed by inverted phase contrast microscope; the cellular activities and ability of proliferation were examined by cell counting kit-8 (CCK-8) method; the ligament fibroblasts related protein expressions including collagen type I, collagen type III, Fibronectin, and Tenascin-C were detected by immunofluorescence staining; specific mRNA expressions of ligament fibroblasts and angiogenesis including collagen type I, collagen type III, Fibronectin, α-smooth muscle actin (α-SMA), and VEGF were measured by real-time fluorescence quantitative PCR. Results The hAMSCs presented monolayer and adherent growth under inverted phase contrast microscope; the flow cytometry results demonstrated that hAMSCs expressed the MSCs phenotypes; the immunofluorescence staining results indicated the hAMSCs had high expression of the vimentin and low expression of CK-19; the hAMSCs possessed the differentiation ability into the osteoblasts, chondroblasts, and lipoblasts. The CCK-8 results displayed that cells reached the peak of growth curve at 7 days in each group, and the proliferation ability in the experimental group was significantly higher than that in the control group at 7 days (P<0.05). The immunofluorescence staining results showed that the expressions of collagen type I, collagen type III, Fibronectin, and Tenascin-C in the experimental group were significantly higher than those in the control group at 5, 10, and15 days after culture (P<0.05). The real-time fluorescence quantitative PCR results revealed that the mRNA relative expressions had an increasing tendency at varying degrees with time in the experimental group (P<0.05). The relative mRNA expressions of collagen type I, collagen type III, Fibronectin, α-SMA, and VEGF in the experimental group were significantly higher than those in the control group at the other time points (P<0.05), but no significant difference was found in the relative mRNA expressions of collagen type I, collagen type III, and VEGF between 2 groups at 5 days (P>0.05). Conclusion The hAMSCs possesses the characteristics of MSCs and good proliferation ability which could be chosen as seed cell source in tissue engineering. The expressions of ligament fibroblasts and angiogenesis related genes could be up-regulated, after inductionin vitro, and the synthesis of ligament fibroblasts related proteins could be strengthened. In addition, the application of TGF-β1 and VEGF could be used as growth factors sources in constructing tissue engineered ligament.
Objective
Through analyzing BKCa channel expression in atrial fibroblasts in patients with sinus rhythm and atrial fibrillation (AF), to explore the mechanism of myocardial fibrosis and provide new therapeutic strategies for the treatment and reversal of AF structure reconstruction.
Methods
We selected 10 patients of rheumatic heart valvular disease who underwent valve replacement surgery. They were 5 patients with sinus rhythm (a sinus rhythm group, 2 males and 3 females with an average age of 49.1±8.3 years) and 5 with AF (an AF group, 3 males and 2 females with an average age of 50.3±5.8 years). About 100 mg tissue was obtained from the right auricula dextra, and the atrial fibroblasts were cultured by tissue block adherence method, and the expression of BKCa channel genes and proteins in cultured fibroblasts was detected by quantitative real-time polymerase chain reaction (qRT-PCR) and western blotting methods.
Results
(1) The general data of 10 patients between the AF group and the sinus rhythm group were compared. There was no significant difference between the two groups in age (t=1.21, P=0.67) and sex (t=2.56, P=0.75). There was statistical difference in the left atrial diameter and the right atrium diameter between the two groups (t=19.45, P=0.01; t=23.52, P=0.06); (2) the mRNA expression of BKCa subunit was detected by qRT-PCR method, and there was no significant difference in the mRNA expression of BKCa α and BKCa β1 between the two groups (t=3.14, P=0.79; t=2.88, P=0.69); (3) the expression of BKCa protein was detected by western blotting method, and there was no significant difference in the protein expression of BKCa α and BKCa β1 between the two groups (t=0.55, P=0.31; t=0.73, P=0.46).
Conclusion
BKCa pathway may not be involved in the pathogenesis and maintenance of AF, but it may play an important role in the process of myocardial fibrosis.
