OBJECTIVE: To explore the autocrine regulation for the transforming growth factor-beta 1(TGF-β1) by the fibroblasts in vitro. METHODS: Fibroblasts were cultured in vitro which isolated from the normal prepuce by circumcision. TGF-β1 concentration were determined with ELISA for the different passages and the different time at the same passage. RESULTS: The TGF-β1 concentration was different for the distinct passage of fibroblasts and achieved the peak (450 ng/L) at the sixth passage. Among the same fibroblasts, the TGF-β1 concentration was different in the various days and passage of the summit (680 ng/L) on the fifth day, which was 2.5 times as high as that of the first day. CONCLUSION: The fibroblasts possess the ability of autocrine of the TGF-β1 and have some regularity. It will provide the theory basis for the research about TGF-β1 regulation mechanism and the medical application about salvia miltiorrhiza.
ObjectiveTo explore if Smad7 protein can inhibit growth of keloids by observing the gene and protein expressions of Smad7, collagen type Ⅰ, and collagen type Ⅲ and cell proliferation after over-expression vectors of Smad7 transfecting keloid fibroblasts (KFb).
MethodsFibroblasts were acquired from 10 male patient with keloids at the age of 20 to 25 years. After in vitro culture, KFb were divided into 3 groups: untransfected group (group A), pcDNA3.1 (-) transfected group (group B), and pcDNA3.1 (-)-smad7 transfected group (group C). The mRNA and protein expression levels of Smad7, collagen type Ⅰ, and collagen type Ⅲ were detected by real-time fluorescence quantitative PCR and Western blot at 48 hours after transfection. The cell proliferation ability was detected by MTT assay at 24 hours after transfection.
ResultsThe relative expression levels of mRNA and protein of Smad7 in group C were significantly higher than those in group A and group B (P < 0.01). The relative expression levels of mRNA and protein of collagen type Ⅰ and collagen type Ⅲ in group C were significantly lower than those in group A and group B (P < 0.01). The relative expression levels of mRNA of collagen type Ⅰ and collagen type Ⅲ in group B were significantly higher than those in group A (P < 0.01); and the relative expression levels of proteins of Smad7, collagen type Ⅰ, and collagen type Ⅲ were significantly lower than those in group A (P < 0.01). The cell proliferation ability in group C was significantly lower than that in group A and group B at each time point by MTT assay (P < 0.05), but no difference was found between group A and group B (P>0.05).
ConclusionGene expressions of collagen type Ⅰ, and collagen type Ⅲ and cell proliferation will be inhibited after KFb are transfected by over-expression vector of Smad7.
Objective To summarize and review the heterogeneity of bone marrow derived stem cells (BMDSCs) and its formation mechanism and significance, and to analyze the possible roles and mechanisms in intestinal epithel ial reconstruction. Methods The related l iterature about BMDSCs heterogeneity and its role in intestinal epithel ial repair was reviewed and analyzed. Results The heterogeneity of BMDSCs provided better explanations for its multi-potency. The probable mechanisms of BMDSCs to repair intestinal epithel ium included direct implantation into intestinal epithel ium, fusion between BMDSCs and intestinal stem cells, and promotion of injury microcirculation reconstruction. Conclusion BMDSCs have a bright future in gastrointestinal injury caused by inflammatory bowl disease and regeneration.
ObjectiveTo explore effect and mechanism of the carcinoma associated fibroblasts (CAFs) of breast cancer on growth and metastasis of breast cancer induced in nude mice by inoculation of CAFs and breast cancer cells.
MethodsBreast cancer cell line of MDA-MB-231 (abbreviated as MDA), CAFs, and normal breast tissue fibroblasts (NFs) of the same breast cancer patient were collected, and mixed with normal saline (NS) or SDF-1 ligand blockers of four nitrogen heterocyclic fourteen alka (AMD3100, abbreviated as AMD) for inoculation of nude mice in vivo. According to the different combination, 36 nude mice were randomly divided into 6 groups:MDA+NS group, NFs+NS group, MDA+NFs+NS group, MDA+NFs+AMD group, MDA+CAFs+AMD group, and MDA+CAFs+NS group. Forty six days after the inoculation and feeding, volume of tumor, metastasis of lymph node, lung or liver were observed. In addition, level of plasma SDF-1 was tested by using ELISA method, and expressions of SDF-1 mRNA and protein in tumor specimens were detected by using real-time PCR and Western blot method respectively.
ResultsExcept for NFs+ NS group, implanted tumor could be seen in nude mice of other 5 groups. In MDA+CAFs+NS group, the volume of tumor[(9.092±2.662) cm3], level of plasma SDF-1[(75.25±16.23) ng/L], and expression levels of SDF-1 mRNA (the median level was 14.714) and protein (the median level was 0.673). of tumor tissue were significantly greater or higher than those of the other 5 groups (P < 0.050). In addition, lymph node metastasis were found in 4 mice in MDA+CAFs+NS group, and 2 in MDA+NS group. The tumor metastasis of lung and liver was not found in all nude mice.
ConclusionsCAFs can promote growth and lymph node metastasis of breast cancer, whose mechanism is related with SDF-1 secreted by CAFs and SDF-1/CXC chemokine receptor 4 (CXCR4), signal pathway.
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.
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.
The aim of this article is to study the effect of tumor necrosis factor alpha (TNF-α) on the expression of matrix metalloproteinases (MMPs) and tissue inhibitors of matrix metalloproteinases (TIMPs) in keratoconus fibroblasts in vitro. Normal cornea and keratoconus fibroblasts were extracted using enzyme digestion method and were cultured in the medium containing TNF-α (0, 10 and 100 ng/mL). The expression of MMPs proteins in the supernatant of corneal fibroblasts and the expression of TIMPs in the normal cornea and keratoconus fibroblasts were detected by Western blot and real-time quantitative polymerase chain reaction respectively. The active form of MMP1 could be detected in the supernatant of keratoconus fibroblasts and upregulated by TNF-α. TNF-α could increase the protein expression of MMP2, MMP3, MMP9 in the supernatant of keratoconus fibroblasts and decrease the gene expression of TIMP1, TIMP2 in keratoconus fibroblasts. The increased MMPs and the decreased TIMPs can increase the degradation of the extracellular matrix. TNF-α may play an important role in the occurrence and development of keratoconus by regulating the expression of MMPs/TIMPs.
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 heat injured keratinocytes (KC) supernatant on the expressions of collagen type I, collagen type III, and matrix metalloproteinase 1 (MMP-1) of dermal fibroblasts (Fb).
Methods
KC and Fb were isolated and cultured. Then the models of heat injured KC and Fb were reproduced in vitro, respectively. The heat injured and normal culture supernatant were collected respectively at 12 hours, and formulated as a 50% concentration of cell-conditioned medium. According to the culture medium, Fb at passage 3-5 was divided into 3 groups. Normal Fb was cultured with the conditioned medium containing 50% heat injured KC culture supernatant (group A), the conditioned medium containing 50% normal KC culture supernatant (group B), and DMEM (group C), respectively. The cells in 3 groups were collected at 24 hours. In addition， the cells in group A were collected at 0, 1, 2, 6, 12, 24, and 48 hours， respectively. Normal Fb was cultured with the conditioned medium containing 50% heat injured Fb culture supernatant. Then, the cells were collected at 0, 1, 2, 6, 12, 24, and 48 hours, respectively. The mRNA levels of the collagen type I, collagen type III, and MMP-1 of Fb were measured by real-time fluorescent quantitative PCR techniques.
Results
At 24 hours after cultured with supernatant of heat injured KC，mRNA relative expression levels of collagen type I, collagen type III, and MMP-1 in group A were significantly higher than those in groups B and C (P lt; 0.05). The mRNA relative expression levels of collagen type I, collagen type III, and MMP-1 in group A gradually increased with time going, showing significant differences between 0 hour and 2, 6, 12, 24, and 48 hours (P lt; 0.05); significant differences were found between different time points after 2 hours (P lt; 0.05). After Fb was treated with supernatant of heat injured Fb, the mRNA relative expression levels of MMP-1 gradually decreased with time going, showing significant differences between 0 hour and 1, 2, 6, 12, 24, and 24 hours (P lt; 0.05); after 2 hours of culture, significant differences were found among different time points (P lt; 0.05).
Conclusion
Heat injured KC supernatant may regulate the mRNA expressions of collagen type I, collagen type III, and MMP-1 of Fb.
Objective To study the biocompatibility of tendon mixedextraction of bovine collagen(tMEBC) and to explore the feasibility of using the threedimensional framework as periodontal tissue engineering scaffold. Methods After being prepared, the tMEBC were cultured with the P4P6 of human periodontal ligament fibroblasts (HPDLFs) in vitro. Threedimensional framework was prepared from bovine tendon. The P4-P6 of HPDLFs (with an initial density of 5×106 cells／ml) were cultured in vitro. Cell attachment andproliferation were measured by cell counting 1 day, 3,5, and 10 days after cell seeding. Histological examination was performed with light microscope and scanning electron microscope 5 and 10 days after cell seeding. Results Porous structure, which supported the proliferation and attachment of HPDLFs, was found in tMEBC. The density of cell increased from 0.556×104 cells／ml 24 hours after cell seeding to 3.944×104 cells／ml 10 days after seeding. Light and scanning electron microscope examinationindicated that HPDLFs were attached and extended on the three-dimensional scaffolds and were well embedded in the newly formed tissue matrix. ConclusiontMEBC has good biocompatibility with the HPDLFs, and can be used as scaffold for cell transplantation in periodontal tissue engineering.
