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Objective:This study aimed to construct a lentiviral expression vector for microRNA-194 and investigate its effect on the metastasis of human osteosarcoma cell line U2-OS. Methods:Pri-and mature miR-194 amplified by PCR were inserted into the plenty-GFP vector and identified by restriction endonuclease digestion and nucleotide sequencing. The osteosarcoma cell line U2-OS was transfected with the lentivirus. Then, the stable transfected cells were used in Transwell and wound healing assay. Results:Restric-tion analysis and sequencing showed that the recombinant lentiviral expression vector was constructed correctly. The titers of obtained overexpression and suppression expression recombinant lentivirus were 1.5*108 and 4*108 TU/ml. Cell metastasis ability was signifi-cantly different in different experimental groups (P<0.01). Conclusion:The lentiviral expression vector for microRNA-194 was suc-cessfully constructed. MicroRNA-194 could influence the metastasis of the osteosarcoma cell line U2-OS;thus, it could be further ex-plored as a potential target in osteosarcoma therapy.
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Objective To identify the specific microRNA (miRNA) that can be taken as a molecular marker for human bone marrow-derived mesenchymal stem cells (MSCs). Methods MSCs were isolated and cultured from bone marrow through density centrifugation and then were induced to differentiate into osteoblasts and chondrocytes. Samples of MSCs, osteoblasts and chondrocytes were detected by miRNA microarrays single channel fluorescence chip to determine the expression levels of miRNAs. Significance Analysis of Microarrays ( SAM, version 2.1 ) software was used to analyze the raw data to determine the miRNAs overexpressed in MSCs, which was validated in the same sample using real time reserve transcriptase polymerase chain reaction (RT-PCR). Results MSCs were successfully isolated from bone marrow and induced to differentiate into osteoblasts and chondrocytes in vitro. Microarrays showed that eight miRNAs (has-miR-424, has-miR-34a, has-miR-593, has-miR-10a, has-miR-148a,has-miR-602, mmu-miR-709 and mmu-miR-665) were overexpressed in MSCs but underexpressed in osteoblasts. Three miRNAs including has-miR-424, PREDICTED_MIR189 and mmu-miR-665, were overexpressed in MSCs but underexpressed in chondrocytes. The has-miR-424 expression in MSCs was 6.6times higher than in osteoblasts and 4.4 times higher than in chondrocytes. The results of real time RTPCR showed that the miR-424 was overexpressed in MSCs, 3.6 times higher than that in osteoblasts and 3.1 times higher than that in chondrocytes, which was coincident with the results of microarray. Conclusions The screened MSCs express more miRNAs in comparison with osteoblasts and chondrocytes,play important roles in maintaining self renewal and undifferentiation of MSCs and is a promising specific molecule marker for MSCs.
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[Objective]To investtgate the specific inhibition of Survivin gene expression by antisense RNA in human osteosarcoma cell line MG63.[Method]Total RNA was extracted from osteosarcoma cell line MG63 cells using Trizol reagent.Coding sequence of Survivin was amplified from MG63 mRNA by RT-PCR and then cloned into inducible eukaryotic vector pMDNA3.After the reducible smmvm antisense vector was construted,MG63 cells were transfected with control pMDNA3 vector or pMDNA3-anti-Survivin and selected by G418.Both of the control and Survivin antisense transfectants were treated with ZnSO_4.These cells cultured on cover slips were observed through immunohisto-chemistry staining,HE staining and electrn microscopy.At the same time,the above cells were cultured and the numbers were counted every other day,thus growth curve was drawn.Apoptosis was analyzed by Annexin V stammg.[Result]cDNA coding Survivin about 420 bp was generated by reverse transcription-PCR Survivin PCR product was cloned reversal into pMDNA3 vector.The MG63 stable tansfectants with either control vector pMDNA3,or pMDNA3-anti-Survivin were established.ZnSO_4 induction of Survivin antistense RNA suppressed the expression of endogenous Survivin mRNA.The cell growth curve showed MG63/pMDNA3-anti-survivm with ZnSO_4 induction proliferated slowly than other kinds of cells(P
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BACKGROUND: At present, there are only morphological observation about the femoral osteolysis in domestic literatures and reports of quantita tive study are also few.OBJECTIVE: To quantitatively analyze the changing status of osteolysis in proximal femur after total joint replacements (TJR) through the changing rules of bone density in proximal end of femur.DESIGN: Observation on patients before and after self-comparison.SETTING: Department of Orthopedics, Tangdu Hospital of Fourth Military Medical University of Chinese PLA.PARTICIPANTS: Twenty-eight hips in 26 patients received total hip replacements in the Department of Orthopedics, Tangdu Hospital of Fourth Military University from March 1994 to March 2004, including 16 hips in 14 male patients and 12 hips in 12 female patients, were selected. Types of prostheses: 17 hips in 16 patients with ameliorated Moore cemented prostheses were taken as cemented prosthesis group with a mean age of 57 years; 11 hips in 10 patients with microbores on the surface of implant were taken as cementless prosthesis group with a mean age of 55 years.Patients were diagnosed as traumatic arthritis before operation with the mean Harris score of 49 (20-77) points on hip. Age, gender and diagnosis after operation in patients of two groups were matched.METHODS: The gray degrees of X-ray in patients of two groups were measured with computer image analyzing, relative values were obtained,I.e. The average gray degrees of 1.0-2.0 cm2 area in femoral trochanter and iliac bone were measured with the same X-ray; Differences between them represented the relative value of bone density in greater trochanter of femur. Changing rules of bone density were quantitatively analyzed.MAIN OUTCOME MEASURES: Relative values of bone density in greater trochanter of femur of patients in two groups one week before and after operation, during the follow-up time were measured respectively.RESULTS: A total of 26 patients (28 hips) with the follow-up time of 9-126months were all involved in the analysis of results. Relative values of bone density were increased to different degree one week after operation. And rate of osteolysis was 100% with a mean value of 57.4 (9-118) in greater trochanter of femur from 17 hips of 16 patients in the cemented group during the follow-up time; Relative value of bone density in greater trochanter of femur in 10 hips of 11 patients in the cementless group during the follow-up time were increased to different degree one week after operation.Rate of osteolysis changing was 100% with a mean value of 72.8 (14-130).Differences between two groups were not significant (P ＞ 0.05). Changes of osteolysis firstly appeared in the 9th month of post-operation and were obvious 2-4 years after operation and lessened in the 6th year.CONCLUSION: There are osteolysis in cemented and cementless prostheses after total hip replacement and no significant differences between two kinds of prostheses.
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BACKGROUND: Patients who suffered total hip replacement are mostconcerned about the survivorship of prosthesis. OBJECTIVE: To evaluate the postoperative curative effect following ce mented and cementless THR with a retrospective method, so as to provideexperience for prolonging the survivoship of prosthesis. DESIGN: Randomized and controlled observation. SETTING: General Center of Orthopaedic Department, General Instituteof Bone Oncology, Tangdu Hospital, Fourth Military Medical University ofChinese PLA. PARTICIPANTS: We admitted 356 patients who underwent THR fromDepartment of Orthopaedics, Tangdu Hospital, Fourth Military Medical U niversity of Chinese PLA between March 1993 and March 2004. Amongthem, 298 were contacted and 105 (108 hips) followed up. The patientsparticipated in the review voluntarily. They were of either gender and haddifferent types of prosthesis. Prosthesis made in China was adopted before2000 and prosthesis made in American STRIKER company after 2000: Prosthesis made in China was made of home-made bone cement; Prosthesisbone cement (import) was provided by American STRIKER prosthesiscompany. Home-made bone cement and import have the same components. Barium was added in both bone cement . The whole operation was con ducted by the physicians who worked in the artificial joint department afterexamination. METHODS: According to informal discussion summary about total hipreplacement of Chinese Journal of Surgery in 1982 and Evaluation Scale ofMayo Total Hip Replacement Curative Effect, we designed follow-up tableby ourselves. Totally 105 (108 hips) patients were followed up, amongthem, 62 (63 hips) were in the cemented group, 43 (45 hips) in the ce mentless group. Pain, function and motion range of the patients and X-raywere evaluated and analyzed respectively. MAIN OUTCOME MEASURES: ① Postoperative pain degrees. ② Postoperative function of hips. ③ Postoperative motion range. ④ width oflight around the prothesis , distance of horizontal or vertical shift of theprosthesis. ⑤ range of ectopic ossification of the prosthesis. ⑥Osteolysisdegree of proximal femur. RESULTS: ①There was no significant difference of lateral femoral painduring follow-up period [Cemented group: 24 hips (38.5%) ,cementlessgroup: 18 hips(40.0% )]. ② Limping appeared in the both two groups ③ There was no significant difference of range of motion above 160° betweentwo groups (Cemented group: 62 hips; cementless group: 44 hips). ④Therewas no significant difference in subsidence of femoral prosthesis and hori zontal or vertical shift of acetabular prosthesis between two groups . ⑤ There was no significant difference of re lative value of femoral proximalbone density between cemented group [57.4(9-118)] and cementless group[72.8( 14-130)]. ⑥There was no significant difference of postoperative cu rative effect, possible survival rate of prosthesis and femoral proximal ex tensive osteolysis of the patients between the two groups. CONCLUSION: Postoperative curative effect of the patients between ce mented group and cementless group are similar, both not obtaining an idealfixed effect. The choice of prosthesis type does not affect the survivorship of prosthesis, but it depends on the age of patients to decide whether rebuilding is necessary or not: Osteolysis is not related to age, gender or prosthesis type of the patients.
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Objective To explore the effects of siRNA on survivin gene expression in osteosarcoma cell line MG63 using siRNA eukaryotic expression vector,and on the cell cycle of osteosarcoma.Method The specific RNA interference(RNAi)fragments targeting survivin gene were synthesized,and they were cloned into pSilencer 3.0-H1 neo plasmid vector.After the vector was constructed,MG63 cells were transfected with negative control vector or RNAi vectors and selected by G418.Expression of protein of survivin in the stable transfected cells was determined by Western blot.These cells which were cultured on cover slips were observed with electron microscopy.Apoptosis analysis was performed with flow cytometry and Hoechst staining.Results Specific siRNA eukaryotic expression vector PsiS targeting survivin gene was constructed successfully.The stable transfectants containing negative control vector and PsiS were obtained.Protein expression of survivin was inhibited significantly in MG63/PsiS cells,whereas survivin gene expression levels were hardly changed in normal MG63 and negative control cells.There were much more pyknotic nuclei found in MG63/PsiS pool than those in MG63 and control.Analysis of DNA content in PsiS transfectants revealed a 7-fold increase in the fraction of sub-G1 peak(apoptotic peak)as compared with other transfectants under the same experimental conditions(P
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Objective To identify the expression of miRNAs in human bone marrow-derived mesenchymal stem cells (hMSCs). Method Low molecular weight RNA fraction (≤200nt) from human bone marrow-derived mesenchymal stem cells was extracted, and polyadenylated by poly(A) polymerase. Then a 5′RNA adapter was ligated to poly(A)-tailed RNA using T4 RNA ligase. RNAs were reversely transcribed and amplified by RT-PCR. The PCR products with a size of about 109 bp were recovered and cloned into pCR 4-TOPO vector. After sequencing, database searching, and expression profiling, the expression of miRNAs in hMSCs was sieved. The expression of novel and some known miRNAs was examined by Northern blotting with small RNAs (≤200nt) isolated from hMSCs, osteosarcoma cell line SOSP-9607 and UMR-106. Results hMSCs were isolated from bone marrow and purified by centrifuge and in vitro culture. Cell markers CD44 were positive, but CD34 and CD45 were negative. A total of 194 clones were subsequently characterized by DNA sequencing and database searching. 52 clones (correspond to 27species) out of 194 clones from hMSCs were identified as miRNAs. One novel miRNAs (PREDICTED-miR-202) was discovered among other 26 known miRNAs, which were predicted in Nature. Northern blotting confirmed that 1 novel miRNAs and 3 known miRNAs (miR-495, miR-34a, miR-17-5p, PREDICTED-miR-202) were stably expressed in hMSCs. Conclusion These findings indicate that a large diverse population of miRNAs are likely important regulators for hMSCs in maintaining their state of self-renewal, and have potential value in stem cells research.