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Objective To investigate the effect of CC chemokine receptor 4(CCR4) on sorafenib radiosensitivity and tumorigenesis of hepatocellular carcinoma cells in nude mouse models.Methods Western blot was used to detect the expression of CCR4 in hepatocellular carcinoma cell line.Lentivirus was utilized to construct PLC/PRF/5 and SMMC-7721 cell lines stably overexpressing and silencing CCR4,which were verified by Western blot.The influence of CCR4 on the radiosensitivity of hepatocellular carcinoma cells was assessed by plate clone formation assay.The effect of CCR4 on the tumorigenesis in hepatocellular carcinoma cells in vivo was evaluated by tumorigenesis assay in nude mice.Results CCR4 was highly expressed in highly-metastatic hepatocellular carcinoma cells and lowly expressed in hepatocellular carcinoma cells with low metastases.The PLC/PRF/5 and SMMC-7721 cells,which stably overexpressed and silenced CCR4,were successfully established.Overexpression of CCR4 reversed the inhibitory effect of sorafenib radiotherapy on PLC/PEF/5,whereas knockdown of CCR4 could increase the radiosensitivity of SMMC-7721 to sorafenib.Overexpressing CCR4 could promote the tumorigenicity of PLC/PEF/5,whereas knockdown of CCR4 could inhibit the tumorigenicity of SMMC-7721 in nude mice.Conclusion CCR4 overexpression significantly reduces the radiosensitivity of PLC/PRF/5 and increases the tumorigenicity in nude mice,whereas knockdown of CCR4 considerably increases the chemosensitivity and radiosensitivity of SMMC-7721 and suppresses the tumorigenicity in nude mice.
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Objective@#To investigate the effect of silencing GRAMD1A and inhibiting STAT5 signaling pathway on the radiosensitivity of Huh7 cells in order to provide new ideas for the clinical combined therapy of hepatocellular carcinoma.@*Methods@# The Huh7 cells silencing GRAMD1A was constructed by infecting lentivirus and verified by qPCR and Western blot. QPCR and luciferase reporter assays were used to detect the effect of silencing GRAMD1A on the expression of STAT5 and its downstream genes. Colony formation and apoptosis were detected to evaluate the effects of silencing GRAMD1A and STAT5 inhibitor SH-4-54 on cell radiosensitivity.@*Results@#After 2 Gy exposure of the constructed Huh7 cells, the colony formation ability of the silencing GRAMD1A combined irradiation group was significantly lower than that of the negative control combined irradiation group, and the difference was statistically significant (t=8. 494, P<0.05). Silence apoptosis in the GRAMD1A combined irradiation group was significantly increased compared with the negative control combined irradiation group (t=3.560, P<0.05). After silencing GRAMD1A, the radiosensitivity of Huh7 cells was significantly increased, and the expression of STAT5 and its downstream genes was significantly reduced in cells.The survival rate of the SH-4-54 inhibitor combined irradiation group was significantly lower than that of the dimethyl sulfoxide combined irradiation group, and the difference was statistically significant (t=8.660, P<0.05). SH-4-54 inhibited STAT5 after passage, the radiosensitivity of Huh7 cells was significantly increased.@*Conclusions@# Silencing GRAMD1A could significantly enhance the radiosensitivity of Huh7 cells via STAT5 signaling pathway, indicating that GRAMD1A plays an important role in the development and progression of HCC. This finding may provide a new target for HCC therapy.
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Objective@#To investigate the effects of inhibiting glutaredoxin 3(GLRX3) expression on proliferation and apoptosis of lung cancer cells.@*Methods@#Western blotting was used to detect the expressions of GLRX3 protein in human embryonic lung fibroblast MRC5 and lung cancer cells, including A427, A549, PC9 and H1299. GLRX3-targeted siRNA (experimental group) and negative siRNA (negative group) were transfected into A549 cells, and the cells without special treatment were blank group. The protein expression levels of GLRX3, cleaved cysteinyl aspartate specific proteinase 3(cleaved caspase-3), signal transducers and activators of transcription 3(STAT3), phosphorylated STAT3(p-STAT3) in each group at 48 hours after transfection were measured by Western blotting. The proliferation ability of differently treated cells at 24 hours, 48 hours and 72 hours after transfection were detected by CCK-8 array. The cell apoptosis at 48 hours after transfection was evaluated by flow cytometry.@*Results@#The protein expression levels of GLRX3 in MRC5, A427, A549, PC9 and H1299 were 0.094±0.010, 0.282±0.021, 0.551±0.045, 0.423±0.039 and 0.454±0.036, respectively. The protein expressions of GLRX3 in tested lung cancer cells were significantly higher than that of MRC5 cells (all P<0.01). The GLRX3 protein expressions in blank group, negative control group and experimental group at 48 hours after transfection were 0.311±0.029, 0.328±0.032 and 0.103±0.012, respectively. GLRX3 protein expression level of experimental group in A549 cells was significantly lower than that of control group (P<0.01), whilewithout statistical difference between the negative group and blank group (P>0.05). A values of experimental group at 24, 48 and 72 hours after transfection in A549 cells were significantly different from those of blank group (all P<0.01). Percent of apoptotic cells in the experimental group was (9.52±0.56)%, which was significantly higher than that of blank group [(1.65±0.22)%] and negative control group [(1.42±0.26)%, all P<0.01]. Consistently, compared with blank group, the cleaved caspase-3 markedly increased in the experimental group (P<0.01). The protein expression of p-STAT3 in the experimental group was significantly lower than the blank group (P<0.01), while no significant difference of STAT3 protein expression was observed among all the groups (P>0.05).@*Conclusions@#Inhibition of GLRX3 gene expression can inhibit the proliferation of lung cancer cells and induce cell apoptosis through up-regulating cleaved caspase-3 expression and down-regulating STAT3 signaling pathway.
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Objective To investigate the effect of Zinc finger E-box binding homeobox protein 1 (ZEB1) on the radiosensitivity of gastric cancer cells AGS and its possible mechanism. Methods AGS cells were irradiated by X-rays at different doses (0, 2, 4, 6, and 8 Gy). Western blot was used to observe the expression of ZEB1 in cells. AGS cells, in logarithmic growth phase, were transfected with of ZEB1 gene or its interference plasmids, the corresponding control plasmids ( pcDNA3. 1 ) and negative control interference plasmids. They were classified as overexpression ZEB1 group, silencing ZEB1 group, control group and negative control group, respectively. The effect of overexpression and silencing ZEB1 on the survival of AGS cells after irradiation were analyzed by colony formation assay. The cell apoptosis rate was analyzed by flow cytometry. The expressions of histone H2A (H2AX), phosphorylated H2AX (γ-H2AX) and telangiectasia mutated gene (ATM) were detected by Western blot. Results The expression of ZEB1 in AGS cells was dependent on radiation dose (F=58. 57, P<0. 05). Overexpression of ZEB1 increased AGS cells viability, inhibitedγ-H2AX expression (t=12. 18, P<0. 05), blocked cell apoptosis (t=7. 27, P<0. 05) and up-regulated ATM expression in time-dependent manner after irradaition (F=165. 70, P <0. 05). Silencing ZEB1 reduced AGS cells viability, increased γ-H2AX expression ( t =12. 88, P<0. 05) and cell apoptosis (t =8. 36, P <0. 05), and down-regulated of ATM expression (F=44. 80, P<0. 05). Conclusions ZEB1 regulates the radiosensitivity of gastric cancer AGS cells by up-regulating ATM expression.
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Objective To translate the English version of the Depression Coping Self-Efficacy Scale (DCSES) into Chinese and test the reliability and validity of the Chinese version of DCSES in patients with depression. Methods Totally 264 patients with depression were recruited and were investigated by the Chinese version of DCSES. Results The total internal consistency coefficient of the Chinese version of DCSES was 0.926, while each dimension were 0.930, 0.908, 0.921, 0.945; split half reliability coefficient was 0.865;the test-retest was 0.752, while each dimension were 0.801, 0.718, 0.729, 0.745, 0.766 (P<0.01);the item-scale correlation ranged from 0.603 to 0.748(P<0.01). Factor analysis got five factors, which explained 58.617% of the total variance. All factor loadings, ranging from 0.447 to 0.762, were statistically significant in the five-factor model and greater than 0.4. Conclusions The Chinese version of DCSES has been proved to be reliable and valid. It can be used as a valid tool for the measurement of the coping self-efficacy in patients with depression.
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Objective To compare the effects of application of clavicular hook plate combined with wire anchors anatomical coracoclavicular ligament reconstruction and application of clavicular hook plate in the treatment of NeerII distal clavicle fracture and Tossy Ⅲtype~V acromioclavicular joint dislocation. Methods A retrospective analysis of the clinical data from June 2006 to June 2013. Total 73 cases patients suffered with Neer Ⅱtype distal clavicle fractures and Tossy Ⅲtype~V acromioclavicular joint dislocation. Of which , 41 cases were subjected to treatment with clavicular hook plate , 32 patients subjected to treatment of using clavicular hook plate combined with anchors .The incision length, operative time, postoperative complications, length of hospital stay and postoperative 1 month, 6 month shoulder VAS score of two groups were analyzed; the shoulder function of both groups after 1 month, 6 months were assessed by using Constant shoulder function assessment method. Results Surgical incision length and operational time between the two groups were statistically significant (P<0.05), while the amount of bleeding was not statistically significant. All patients were followed up . The two groups did not occur any complications such as loosening, decoupling, acromioclavicular joint dislocation and wound infections. Hospitalization time was 5~14 d (averaged 10d), no significant difference between two groups. 4 the shoulder Constant score and VAS scores showed no significant difference 1 months postoperation; 6 months after hook plate removed, VAS score and Constant shoulder score improved significantly in anchors hook plate group (P<0.05). Conclusion Anatomical coracoclavicular ligament reconstruction by application of hook plate combined with anchors is a good biomechanical model characterized with simple surgery , less trauma and good clinical outcomes , worthy of clinical application.
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BACKGROUND:Knee joint function limitation often occurs after internal fixation of complex femoral condyle fractures, but the mechanism and its influencing factors are also unclear. OBJECTIVE:To screen and analyze the relevant factors of knee joint function limitation after internal fixation of complex femoral condyle fractures. METHODS:We retrospectively summarized postoperative fol ow-up data of 6 and 12 months from 121 patients with complex femoral condyle fractures. Knee joint function recovery was evaluated according to Merchan criteria. A multiple stepwise regression analysis was carried out in terms of gender, age, causes, concomitant injuries, skin and soft tissue injury, fracture type, fixed method, operation time, postoperative plaster fixed situation, healing of postoperative incision, bone healing and postoperative functional exercises, to summarize the relevant influencing factors for knee joint function limitation. RESULTS AND CONCLUSION:Whether the knee joint function after internal fixation was limited acted as the dependent variable Y, and factors with statistical significance of the single factor analysis served as the independent variable X. We used the multiple stepwise regression analysis for multiple factors analysis. Results showed that the gender of patients (X1), with or without concomitant injuries (X3), soft tissue damage (X4) and operation time (X6), a total of four factors, could not be introduced into the model, suggesting that these four factors had no significant correlation with postoperative knee joint function limitation. Another eight factors could be introduced into the factor analysis model, showing that the cause of injury (X2), fracture type (X5), the choice of internal fixation (X7), with or without bone graft (X8), with or without postoperative plaster cast (X9), postoperative knee joint functional exercise or not (X10) and postoperative wound healing (X11), the degree of postoperative bone healing (X12) are closely related to postoperative knee joint function limitation in complex femoral condyles fractures.
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BACKGROUND: Now, dual-energy X-ray absorptiometry is international y recognized as gold standard for the diagnosis of osteoporosis, but the errors can be found in the measurement results due to the heterotopic ossification and bone hyperplasia exists in the measurement part. OBJECTIVE: To investigate the clinical significance of bone metabolic markers in the diagnosis and treatment of elderly patients with osteoporotic fractures, and to research its correlation with the changes of pathological histology and bone mineral density. METHODS: Four bone biochemical markers in 50 elderly patients with osteoporosic fractures were measured preoperatively. According to the results, 25 patients had significantly increased tartrate-resistant acid phosphatase 5b (considered as the increased tartrate-resistant acid phosphatase 5b group), and 25 patients had increased bone alkaline phosphatase (considered as the increased bone alkaline phosphatase group). During operation, the bone tissues of eight patients in each group were treated with hematoxylin-eosin staining and electron microscopy scanning in order to detect the pathological changes. After operation, the patients in the increased tartrate-resistant acid phosphatase 5b group received salmon calcitonin anti-osteoporosis treatment, and the patients in the increased bone alkaline phosphatase group received the anti-osteoporosis treatment of bone peptide injection. The bone mineral density and the four bone biochemical markers were detected again at 6 months after treatment. RESULTS AND CONCLUSION: There were no significant differences in the preoperative bone mineral density and four biomechanical markers between two groups (P > 0.05). The pathological examination results of bone tissue on the fracture site showed that the number of osteoblasts was reduced and the number of oeteoclasts was increased in the increased tartrate-resistant acid phosphatase 5b group; while in the increased bone alkaline phosphatase group, the pathological examination results showed the number of osteoblasts was reduced; the trabecular bone/bone area ratio was decreased in two groups, and there was a significant difference in the decrease degree between two groups (P < 0.05). The electron microscope scanning showed that the osteoclasts of two groups were more active than that of the normal group. The sloppy of trabecular bone in the increased tartrate-resistant acid phosphatase 5b group was more obvious than that in the increased bone alkaline phosphatase group, and the absorption vacuoles were increased. There were significant differences in the bone mineral density and four biomechanical markers between two groups before and after anti-osteoporosis treatment (P < 0.05). The detection of bone metabolic markers could help us to make it clearly that the main function of osteoblast reduce or osteoclast increase in bone tissue of patients, and guide us to use anti-osteoporosis drugs in target. Pathological histology examination can better reflect the condition of osteoblasts, osteoclasts and trabecular bone in bone tissue on the fracture site. Target application of anti-osteoporosis drugs in the osteoporosis patients can effectively improve the efficacy and reduce the relative complications.
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BACKGROUND:Adipose-derived stem cel s and bone marrow mesenchymal stem cel s are used widely in cartilage tissue engineering, and there are many similarities in biological characteristics between two kinds of cel s. OBJECTIVE:To compare the chondrogenic potential of bone marrow mesenchymal stem cel s and adipose-derived stem cel s in vitro. METHODS:Adipose-derived stem cel s were isolated from the 3-month-old New Zealand white rabbits’ abdomen. Bilateral femurs of rabbits were obtained, and then the bone marrow mesenchymal stem cel s were separated with the adherence screening method. The growth curve of the passage 3 adipose-derived stem cel s and bone marrow mesenchymal stem cel s were drawn, and the doubling time of two kinds of cel s was compared. Then the passage 3 adipose-derived stem cel s and bone marrow mesenchymal stem cel s were treated with chondrogenic induction. After induced for 14 days, the adipose-derived stem cel s and bone marrow mesenchymal stem cel s were treated with toluidine blue staining and type Ⅱ immunohistochemistry staining respectively. RESULTS AND CONCLUSION:Primary bone marrow mesenchymal stem cel s showed aggregative growth, while the primary adipose-derived stem cel s were in single and scattered growth. The proliferation speed of adipose-derived stem cel s was faster than that of bone marrow mesenchymal stem cel s, while the doubling time of adipose-derived stem cel s was shorter than that of the bone marrow mesenchymal stem cel s. After chondrogenic induction for 14 days, both adipose-derived stem cel s and bone marrow mesenchymal stem cel s could express glycosaminoglycans and type Ⅱcol agen, and the expression level of type Ⅱ col agen in bone marrow mesenchymal stem cel s after chondrogenic induction was higher than that in the adipose-derived stem cel s. The in vitro proliferation of adipose-derived stem cel s and bone marrow mesenchymal stem cel s was rapid and stable, but the proliferative ability of adipose-derived stem cel s was faster than that of bone marrow mesenchymal stem cel s. When cultured in single layer, both adipose-derived stem cel s and bone marrow mesenchymal stem cel s could transform into chondrocytes under certain conditions, but bone marrow mesenchymal stem cel s seemed to be more potential than adipose-derived stem cel s.
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BACKGROUND:There are reports concerning differentiation of adipose-derived mesenchymal stem cells(ADSCs)into chondrocytes using gene transfection technique.However,the transfection of adenovirus and adeno-associated virus into ADSCs is various.It is controversial whether adeno-associated virus(AAV)can transfect ADSCs.OBJECTIVE:To observe the enhanced green fluorescent protein(EGFP)expression following Ad5-EGFP and rAAV2-EGFP transfection into ADSCs,and investigate the cell proliferation ability following transfection.METHODS:ADSCs were isolated from the adipose tissue,which was from 6-month-old New Zealand albino rabbit back and neck by mechanical digestion and enzyme digestion,then ADSCs were cultured and amplified in vitro.ADSCs were infected with Ad5-EGFP and rAAV2-EGFP,and the EGFP expression was observed.A total of 2 μL sodium butyrate(1 mol/L)was added into the medium after rAAV2-EGFP transfection.MTT assay was used to detect the gene transfection effects on reproductive activity of ADSCs.RESULTS AND CONCLUSION:ADSCs isolated and cultured in vitro were flat,long-spindle and amplified stabry.The cell morphology was uniform.Many green fluorescent cells were observed in Ad5-EGFP and rAAV2-EGFP groups.Transfection efficiencies were about 88% and 83%.Adenovirus and adeno-associated virus vector can be transfected with ADSCs,and transfection efficiency is high.Adeno-associated virus needs sodium butyrate to increase its level of gene expression.
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[Objective]To investigate the methods of preparing acellular cartilaginous matrix(ACM) and constructing the tissue engineered cartilage with adipose-derived stem cells(ADSCs)-seeded acellular cartilaginous matrix in vitro.[Method]The ADSCs were isolated from adult New Zealand albino rabbits by collagenase,cultured and amplified in vitro.Fresh cartilage isolated from adult New Zealand albino rabbits were freeze-dried for twelve hours and then treated with Triton X-100,Dnase and RNase so as to obtain the ACM.After sterilized with ultra-violet,the ADSCs were seeded in the acellular cartilaginous matrix at a final density of 2?107/L and cultured in chondrogenic differentiation medium for two weeks in order to construct tissue engineered cartilage.Histology,immunohistochemistry and transmission electron microscope(TEM) were applied to examine the fresh ACM and tissue engineered cartilage.[Result]The test of hematoxylin-eosin(HE) staining and TEM showed no cellular structure in the ACM with only recesses left by removed cells.The ACM had suitable interval porosity and aperture size.After integrated with ADSCs,cells migrated into the ACM and adhered to the surface of material and grew well.After cultured in chondrogenic differentiation medium for two weeks,immunohistochemical staining of type Ⅱ collagen showed part of the cells in the material had enhanced expression of type Ⅱ collagen.[Conclusion]ACM can be used as scaffold material in cartilage tissue engineering.If it was seeded with ADSCs and cultured in chondrogenic differentiation medium,ACM can be used to construct cartilage tissue successfully.
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AIM: To explore the role of TGF-?_1 and signaling transduction molecule, Smad4, in the development of glomerulosclerosis. METHODS: Expression levels of TGF-?_1, Smad4, collagen Ⅰ proteins were evaluated by immunohistochemistry in renal biopsies from 38 cases with a spectrum of glomerulonephritis, and compared with 20 normal kidney tissue with image analysis system. After stimulation with TGF-?_1, expressions of endogenous Smad4 and collagen Ⅰ mRNA and proteins and its modulation by TGF?_1 were evaluated by RT-PCR and Western blotting analyses in cultured human mesangial cells. RESULTS: All types of proliferative and sclerotized glomerulonephritis showed an increased expression of TGF-?_1, Smad4 and collagen Ⅰ ompared with the 20 normal kidney tissue in glomerular (P
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By using of combined HRP retrograde tracing and immunocytoche mistry methods, the projection from the caudal part of the ventrolateral medulla (VLM) to the nucleus accumbens (Acb) was examined. When HRP was injected into the ventral and medial area of the caudal part of Acb, the labeled cell bodies were mostly found in the bilateral (predominantly ipsilateral) caudal part of the VLM. When HRP technique (HRP injected into the Acb)was combined with immunocytochemical method, many HRP-TH, HRP-NT and HRP-CCK double labeled cell bodies were found in the VLM. The number of the HRP-TH double labeled cell bodies were numerous, while HRP-NT and HRP-CCK doublelabeled cells were less. HRP-TH double labeled neurons were also found in the reticular formation between the solitary tract nucleus and VLM.