Objective To explore the proper dosage of establishment of stable hepatic oval cells （HOC） prolif-eration model by using 2-acetaminofluorene （2-AAF） combined with two-third partial hepatectomy （2/3 PH） surgery， and to explore isolated and cultured method of HOC in vitro. Methods The 174 Wistar rats were randomly divided into 4 experimental groups （each group enrolled 30 rats）， saline group （n=30）， and untreated group （n=24）. Rats of 4 experi-mental groups were underwent gavage of 5， 10， 15， and 20 mg/（kg ? d） 2-AAF， corresponding to the groups from No.1 to No.4 group. Rats of saline group received saline gavage and rats of untreated group didn’t received any treatment. A standard 2/3 PH surgery was performed on the 5th day after gavage， then the same gavage method was still administrated as preoperation untill rats were sacrificed. The liver tissues of 6 selected rats were adopted and identified by HE staining and immunohistochemical staining on 4， 8， 12， and 16 days after PH for observation of the proliferation of HOC in every group， on 4 days， levels of alanine aminotransferase （ALT） and aspartate aminotransferase （AST） were tested in addition. HOC were isolated and purified by collagenase perfusion method and percoll gradient centrifugation. Results The surv-ival rates of untreated group，saline group，No.1 group，No.2 group，No.3 group，and No.4 group were 100% （24/24），93% （28/30），93% （28/30），90% （27/30），90% （27/30），and 80% （24/30） respectively. Compared with the saline group and untreated group， the levels of serum ALT and AST increased significantly in No.2， No.3， and No.4 group on the 4th day after PH （P＜0.05）. The results of HE staining showed that No.2， No.3， and No.4 group were observed visibly different level of damage at liver tissue， and the proliferation level of HOC were most obviously in No.3 and No.4 group. The results of immunohistochemical staining revealed that proliferation cells were positively expressed oval cell marker-6 （OV-6）. The number of OV-6 positive cells were increased significantly with the increase of dosage of 2-AAF between 4 days and 12 days after operation， and proliferation levels were related with dosages of 2-AAF （P＜0.05）. In all cultured cells， 80% of cells were OV-6 positive cells after isolation and culture by using collagenase perfusion method and percoll gradient centrifugation. Conclusions The methods of gavage of 2-AAF at 15 mg/（kg ? d） combined with 2/3 PH surgery can establish the HOC proliferation model on the 12th day， as well as the rats have lower mortality and better tolerance， especially. The collagenase perfusion method and percoll gradient centrifugation can be used to isolate HOC effectively.
Objective To investigate the effects of different temperatures on the system of in vitro physiological environment fostering limbs. Methods Twenty-four limbs were harvested from 6 adult Bama mini pigs and were randomly divided into 4 groups (n=6) according to different temperatures: limbs were placed in in vitro physiological environment foster-ing limbs at 26℃ (group A), 4℃ (group B), 10℃ (group C), and 18℃(group D). After 12 hours of perfusion, the morphology observation was done for the structure and ultrastructure changes of the skeletal muscle by light microscope and transmission electron microscope. The mRNA levels of tumor necrosis factor α (TNF-α) and interleukin 1β (IL-1β) were detected by real-time fluorescent quantitative PCR (RT-qPCR). Results Histological results showed that the skeletal muscle exhibited mild edema, integrity of the sarcolemma, and occasional perivascular inflammatory cell infiltration in groups B, C, and D, meanwhile, the cells of group C had normal morphology; however, muscle fibers degenerated, muscle cells were seriously damaged, a great number of inflammatory cells infiltrated in the fractured muscle fibers in group A. Transmission electron microscope results showed as follows: the muscle fibers arranged in disorder, and many focal solubility necrosis occurred in group A; the muscle fibers arranged in order relatively and sarcolemma was still intact, with mild swelling and flocculent degenerative mitochondria in group B; a large number of muscle fibers arranged in order and regularity with clear sarcomere in group C; and the muscle fibers arranged in disorder and irregularity and partly dissolved in group D. RT-qPCR results showed that the expressions of inflammatory factor TNF-α and IL-1β mRNA in group A were significantly higher than those in groups B, C, and D (P lt; 0.05); the expressions were significantly lower in groups B and C than in group D, and in group C than in group B (P lt; 0.05). Conclusion In the system of in vitro physiological environment fostering limbs, temperature plays an important role in the preservation of amputated limbs. It is suggested that 10℃ can significantly attenuate the reperfusion-induced skeletal muscle cell injuries in this system.
【Abstract】 Objective To reconstruct three different kinds of tympanic membrane in vitro by tissue engineeringtechnique, and to examine their histological structures and mechanical properties. Methods The skin and dura of pig(weight 30 kg) were processed with high satuated sal ine and enzymes to make extracellular matrix. Meanwhile, fibroblasts(1×106 /mL,0.2 mL) were seeded on the surface of these two scaffolds and collagen. The composite tissues were cultured in vitro for 1 week and examined in histological structure and mechanical properties. Results Fibroblasts cultured were spindle–shaped and could grow and attach to these scaffolds with a arrangement of sarciniform and parallel. The reconstructed tissue of ECM and collagen appeared to integrate well and had better bio-compatibil ity. The mean thickness of the collagen, the skin and the dura (all covered with fibroblasts) were 9.4, 10.0 and 10.4 μm respectively. The tension of the collagen was (1.417±0.030) N/mm2, of the acellular dermal matrix was (24.500±2.040) N/mm2(being close to the tension of normal tympanic membrane, 26.700 N/ mm2),of the acellular dura was (53.300±2.600) N/mm2. Conclusion The results suggest that the tension and the thinkness of acellular dermal matrix is similar to the normal tympanic membrane of guinea pig, it is an ideal material for tympanoplasty.
ObjectiveTo evaluate the feasibility and security of endovascular repair of abdominal aorta using branched stent graft in a novel in vitro vascular model. MethodsThe branched stent graft for the abdominal aorta was designed. The novel in vitro vascular model was established to test this stent graft. Attempts were made to optimize the procedure of stent graft and to evaluate the feasibility of this device. The branched stent graft for abdominal aorta was tested by a novel in vitro vascular model. The number of stent graft released and expanded was recorded respectively. The pressure and situation of branch vessels were assessed before and after stent graft released. The endoleak during releasing process was observed by digital subtraction angiography (DSA). ResultsThe stent graft was successfully deployed in the novel in vitro vascular model. The releasing process was all properly achieved (100%, 30/30). The pressure changes of branch vessels were no statistical significances (P > 0.05) between before and after stent graft released. The stent grafts were well landed, and were fully expanded and properly positioned by DSA. No endoleak occurred. ConclusionThe branched stent graft for abdominal aorta in a novel in vitro vascular model is safe and feasible.
Objective To investigate the role of anti apoptosis gene bcl-2 in the survival of cultured uveal melanoma UM cells. Methods Antisense oligodeoxynucleotides (AS-ODN) bcl-2 were delivered with cationic lipid to primary cultured UM cells. The inhibitory effect of AS-ODN bcl-2 on proliferation of UM cells was examined by 3,-4,5 Dimethyliazol-2,5 diphenyl tetrazolium bromide (MTT) method. Using DNA ladder to determine if the UM cells had been apoptotic. Bcl-2 expression was detected by RT-PCR and Westernblot technics. Results The effect of AS-ODN bcl-2 in inhibiting the proliferation of cultured UM cells had opposite relation to dosage. It down regulated the mRNA and protein level of bcl-2 gene, and the sense ODN didn′t have this effect. Conclusion AS-ODN bcl-2 can down regulate bcl-2 expression, inhibits UM cells proliferation and induces apoptosis. (Chin J Ocul Fundus Dis, 2002, 18: 38-41)
【Abstract】 Objective To observe the effect of cytokines and combinations in the inducement of human umbil icalcord blood-derived CD34+ cells into hepatocyte-l ike cells. Methods The mononuclear cells (MNCs) were derived by density gradient centrifugation and the CD34+ cells were sperated from MNCs. The human umbil ical cord blood-derived CD34+ cells were cultivated through 49 different combinations of cytokines including leukemia inhibitor factor (LIF), oncostatin M, bFGF, aFGF, hepatocyte growth factor, EGF and stem cell factor for 28 days, and the concentrations of the cytokines were 10, 10, 10, 10, 20, 20 and 50 ng/mL, respectively. The mRNAs of cytokeratin 19 (CK-19), CK-18, glutamine synthetase (GS), human albumin (ALB) and α-fetoprotein (AFP) were detected every seven days. The ALB secretion abil ity, detoxification abil ity and hepatin synthesis abil ity of the induced cells were detected by immunofluorescence assay, indocyanine green (ICG) and periodic acid-schiff assay, respectively. The fresh umbil ical cord blood-derived CD34+ cells were detected at the same time as a control. Results The mRNAs of CK-19, CK-18 and GS could be transcribed in all the induced cells, but the transcription of the mRNAs of ALB and AFP which was the special mark of mature hepatocyte and l iver stem cell, respectively, was not found. All the mRNAs could not be found in freshly isolated umbil ical cord blood-derived CD34+ cells. All the cells induced in vitro could not release ALB, and not help the detoxification of ICG which was the fundamental function of mature l iver cells. These results were the same in the control group. The hepatin synthesis abil ity of all the induced cells increased by comparison to the fresh ones. Conclusion Though some mRNAs of proteins which are transcribed in hepatocytes can be found in the induced cells, umbil ical cord blood-derived CD34+ cells could not be transdifferentiated into hepatocyte-l ike cells through cytokines in vitro.
Objective To observe the morphology and growing status of mesenchymal stem cells(MSCs) of human bone marrow in vitro, in order to confirm that MSCs of human bone marrow are ideal seed cells and provide basic theory for further MSCs research. Methods The methods of density gradient centrifugation with lymphocyte separation medium for human and adherent filtration were used to isolate and purify MSCs of human bone marrow. We observed the cellular growth status and morphology of the primary MSCs and the surface antigens of second passage MSCs were tested. Results The primary culture cells fused into monolayer after 14-16 d. The passage cells kept the same morphological characteristics of primary culture cells. Ultrastructure of the second passage MSCs showed that the shape of nuclei was irregular, there were multiple nucleoli in some of the nuclei, and morphological differentiation of intracytoplasm organelles was immature. The growth curve of the first, fifth and tenth passage cells showed a logarithmic growth at day 3, a peak growth at day 5, and no clones occurred after tenth passage. Cloning efficiency of first passage, fifth passage and tenth passage was respectively 25.83%±2.93%, 14.67%±1.63% and 4.67%±0.52%. Test of MSCs phenotypic characteristics showed a high homogeneity among the cells and surface antigen profiles were positive for CD29, CD44 and negative for CD34, CD45. Conclusion The methods of density gradient centrifugation with lymphocyte separation medium for human and adherent filtration are simple, economic and efficient to isolate and purify MSCs from human bone marrow. With a high proliferating ability in vitro, MSCs from human bone marrow are ideal seed cells for tissue engineers.
ObjectiveTo compare the clinical outcomes of different pituitary down regulation protocols with gonadotropin-releasing hormone agonist (GnRH-a) in patients undergoing in vitro fertilization and embryo transfer (IVF-ET) treatment. MethodsThe clinical data of 358 IVF cycles in women at 40 years old or younger from November 2012 to January 2013 in the West China Second University Hospital were analyzed retrospectively. All the 358 cycles were divided into two groups, according to whether the leading follicle diameter was <14 mm (group A, 158 cycles) or ≥14 mm (group B, 200 cycles) after discontinuing the GnRH-a. The clinical outcomes were compared between the two groups. ResultsCompared with group B, the amount of gonadotropins used was significantly more, and the time of gonadotropin use was also significantly longer in group A (P<0.05). However, the serum level of estradiol (E2), progesterone (P) and Luteinizing hormone (LH), incidence of premature P rise, retrieved ovum number, the rates of implantation, clinical pregnancy, miscarriage and live birth did not significantly differ between the two groups (P>0.05). ConclusionDiscontinuing the use of GnRH-a in early stage of controlled ovarian stimulation can keep effective pituitary down regulation and it has the same optimal clinical outcomes in patients undergoing IVF-ET.
Human SW480 colonic cancer cell line was evaluated for its growth response to pentagastrin, gastrin receptor antagonist proglumide (PGL) in vitro by MTT assay and flow cytometry. The results showed that gastrin possessed a proliferative effect on SW480 cell, PGL alone had no obvious effect on SW480 cell, but it inhibited gastrin-induced growth of SW480 cell with dosage dependent when it was used with gastrin, its inhibitive effect did not steadly increase at a dose＞32μg/ml. This suggests that effect of gastrin is achieved via gastrin receptor. Gastrin promoted the sythesis of DNA, protein and triggered the cancer cell shifting from phase G0/G1 to phase S, G2M. PLG inhibited the effect of gastrin, it suggests that gastrin possessed a proliferation on SW480 cell at post receptor is achieved by the effect of gastrin on cell cycle.
Objective To establish a rapid, simple, and economic method to prepare osteoporosis (OP) in vitro model. Methods Eighty pairs of fresh goat femur were collected from 18-month-old female goats and were randomly divided into 4 groups (20 pairs in each group). The femur was immersed decalcifying solution (18% EDTA) for 1-5 days (group B), 6-10 days (group C), and 11-15 days (group D), while group A had no treatment as control. Four pairs of femur were taken out every day. Quantitative computed tomography was used to scan the medial and lateral femoral condyles, and the bone mineral density (BMD) was calculated. Electronic universal testing machine was used to do three-point bending test and compress and tensile ultimate strenght test, and the mechanical parameters for femur were calculated. Results With demineralized time passing, BMD of the medial and lateral femoral condyles were downtrend in groups A, B, C, and D, showing significant differences among 4 groups (P lt; 0.05); BMD of the lateral femoral condyle was significantly higher than that of the medial femoral condyle in each group (P lt; 0.05). The three-point bending test showed that broken load, ultimate strength, and elastic modulus of groups A and B were significantly higher than those of groups C and D (P lt; 0.05); but no significant difference was found between groups A and B, and between groups C and D (P gt; 0.05). Compress and tensile ultimate strength test showed that the compress and tensile ultimate strengths were significantly higher in group A than in groups C and D (P lt; 0.05), and in group B than in group D (P lt; 0.05), but no significant difference was found between groups A and B, between groups B and C, and between groups C and D (P gt; 0.05). Conclusion The 18% EDTA immersing for 6-15 days is a fast, simple, economical method to prepare an OP in vitro model of goat femur.