BACKGROUND: Excessive apoptosis of ovary granulosa cell is a dominant cause of premature ovarian failure and bcl-2 gene is able to inhibit cell apoptosis. But studies demonstrate that,the guanine and cytosine (GC) content reaches 60% in the rat bcl-2 gene sequence. This gene cannot be amplified using routine polymerase chain reaction method. OBJECTIVE: To clone and identify the bcl-2 gene riched GC. DESIGN,TIME AND SETTING: Open experiment was finished in the Laboratory of Medicine and Molecular Biology,Life Science School of Sun Yat-sen University from May to December in 2007. MATERIALS: Wistar rats were purchased from Experimental Animal Center of Sun Yat-sen University. Ecoli DH5?was preserved by Laboratory of Medicine and Molecular Biology,Life Science School of Sun Yat-sen University; pMD18-T vector was purchased from Takara Biotechnology (Dalian) Co.,Ltd. METHODS: The bcl-2-cDNA,in which GC accounted for 60.6%,was obtained by modified reverse transcription-polymerase chain reaction from kidney tissue of Wistar rats,and was cloned into vector-pMD18-T. Characterizations and sequencing of the pMD18-T-bcl-2 were carried out by polymerase chain reaction screening of individual bacterial colonies. MAIN OUTCOME MEASURES: Cloning and purification of bcl-2 gene cDNA; results after connecting bcl-2 gene cDNA to pMD18-T vector and transducting Ecoli DH5?,identification of positive clone and results of sequencing. RESULTS: The bcl-2 gene was identified by the clone and DNA sequencing. DNA sequence analysis was consistent with Genebank sequence,with a 99% homology. CONCLUSION: The gene riched GC is difficult to be amplified,bcl-2-cDNA can be cloned and constructed into cloning vector pMD18-T successfully by the efficient technique for other genes riched GC.

Objective To clone the full-length cDNA of rat B cell lymphoma-2(bcl-2) gene,then construct and identify the cytomegavirus-mediated lentiviral expression vector of bcl-2 gene,and assess the gene expression in 293T cell,which is a human embryonic kidney cell line.Methods The full-length bcl-2-cDNA fragment was amplified by reverse transcription polymerase chain reaction(RT-PCR) from the kidney tissue of a Wistar rat.The double-stranded oligonucleotides(dsOligoe) were then cloned into the pMD18-T plasmid.After confirmation of a correct construction by sequencing,the positive clone was subcloned into pGC-FU vector with enhanced green fluorescent protein(EGFP),and then transformed into DH5a competent cells.The restricted endonuclease and T4 DNA ligase were used to construct the lentiviral expression vector plasmid pGC-FU-bcl-2 which,combined with the lentiviral packing materials(pHelper 1.0,pHelper 2.0),was then transfected into 293T cell line to form the recombinant lentivirus pGC-FU-bcl-2,and it was used to transfect the 293T cells.The expression of pGC-FU-bcl-2 was further verified by detecting EGFP and bcl-2.Results 1) It was verified by DNA sequencing that the sequence of rat bcl-2 gene was consistent with reported sequence in GenBank.2) The bcl-2 gene was successfully combined in pGC-FU-bcl-2 recombinant plasmid which could be transfected into human embryonic kidney cells.3) The recombinant virus pGC-FU-bcl-2 could be obtained from the 293T cells by co-transfection of pGC-FU-bcl-2 and packing plasmids.4) Targeting gene could be cloned into 293T cells by the recombinant lentivirus with steady expression.The fluorescent protein could be observed under microscope and the expression of bcl-2 protein was detected by Western blotting.Conclusions The lentiviral expression vector containing EGFP and bcl-2 gene has been successfully constructed,with which the transfected 293 T cells can lead to a steady expression of bcl-2 protein.The present study provides a basis for the further study of the function of bcl-2 gene and a potential therapy for the diseases relating to apoptosis.

Objective To determine the minimum alveolar concentration(MAC)of sevoflurane for blunting the responses to removal of the laryngeal mask airway(LMA)in 50%anesthetized children.Methods Twenty-five ASA Ⅰ or Ⅱ children aged 3-8 yr undergoing elective surgery under general anesthesia weTe enrolled in this stuay.Anesthesia was induced with inhalation of 8%Sevoflurane.LMA was inserted when the children lost eyelash reflex and the lower jaw was relaxed.Anesthesia was maintained with 3%sevoflurane.All the children kept spontaneous breathing during operation.Assisted ventilation waw performed when necessary to maintain PET CO2 at 35-45 mm Hg.After the surgery the target end-tidal sevoflurane concentration was maintained for 10 min before LMA was removed.Up-and-down sequential allocation was used to determine山e MAC.The initial end-tidal concentration was 1%and was increased/decreased by 20%in the next patient if the extubation response was positive or negative.Limb movement,breath-holding,laryngosposm and hypoxemia(SpO2<95%)were considered to be the signs of positive response.The midpoint from positive response to negative response was made the balance point.and the mean value ofthe concentrations of sevoflurane at all the balance points were calculated as MAC.Results The end-tidal sevoflurane concentration for blunting the responses to removal of LMA was 0.98%.Conclusion The MAC of sevoflilrane for blunting the responses to removal of LMA in 50%anesthetized children(aged 3-8 yr)is 0.98%.

BACKGROUND:Three-dimensional (3D) biological printing uses tissue engineering and stem cel research results, and takes living cel s and other cel active ingredients as printing materials, final y realizing biological tissue printing and production. OBJECTIVE:To review the application and research progress of 3D printing in the field of orthopaedics. METHODS:A computer-based search of PubMed, Ovid, and CNKI databases was performed for relevant literatures about application and research progress of 3D printing in the field of orthopaedics, al of which were published from 2007 to 2016.“three-dimensional printing, 3D printing, plastic and reconstructive surgery, orthopaedic, organ printing”were used as keywords during the searching process. According to inclusion and exclusion criteria, 44 articles were included for further analysis and summary. RESULTS AND CONCLUSION:3D printing was mainly applied into craniomaxil ofacial reconstruction, nose, ear and cartilage reconstruction, breast reconstruction, and skin printing. Its application in bone and prosthetic fabrication was quite mature. Based on the development of 3D printing from prosthetic fabrication to bioactive printing, organ printing wil eventual y become reality to completely solve the autologous or al ograft transplantation limitations.

Objective To investigate the expression of high mobility group box 1 (HMGBI) of TLR4 endogenous ligand and distant organ tissue injury after intestine ischemia/reperfusion and drainage of lymph fluid in rats. Methods Twenty-four Sprague-Dawley (SD) male rats (SPF grade) were evenly divided into 3 groups:Sham surgery group,intestine ischemia-reperfusion (I/R) group,and intestine ischemia-reperfusion with drainage of intestine lymph fluid (IR + drainage) group.The injury of distant organs such as lungs,liver,kidney was evaluated;The expression of high mobility group box 1 (HMGBI) of TLR4 endogenous ligand in intestine,lung and liver after the ischemia-reperfusion injury was measured by immunohistochemistry.Result HE stained sections,as well as HMGB1 immunohistochemistry results showed that the injury of ischemia/reperfusion (I/R) group and ischemia/reperfusion (I/R) + drainage group were more severe than that in the sham group.A large number of cells stained in I/R group,indicating that HMGB1 expression increased.The injury in I/R + drainage group was significantly less severe than I/R group.Western blot tests showed that the expression of HMGB1 in jejunum,ileum,liver,lung increased significantly in I/R group after L/R injury.Gray-scale values of HMGB1/β-actin were 0.3145 ± 0.0549、 1.7352 ± 0.3280、1.4443 ± 0.0926、3.1382 ± 0.4202.Lymph drainage significantly alleviated the damage,the expression of HMGB1 were significantly lower (P ＜0.05).Gray-scale values of HMGB1/β-actin were 0.1745 ± 0.0327、 1.1083 ± 0.2098、 1.1862 ± 0.1221、2.1095 ± 0.1993. Conclusion Increased expression of HMGB1 of TLR4 endogenous ligand is associated with intestinal and distant tissue injury during intestinal ischemia-reperfusion injury.Drainage of lymph fluid can block the gutlymph pathway and thus reduce the source of HMGB1 from the intestinal as well as the injury of distant tissue.

Seven guaiane-type sesquiterpenoids, a new compound 6-formyl-5-isopropyl-3-hydroxymethyl-7-methyl-1H-indene (1), a new natural product 5-isopropyl-3, 7-dimethyl-1H-indene-1-one (2), along with five known compounds: guaiazulene (3), 4-formyl-7-isopropyl-10-methylazulene (4), sesquiterpene ketolactone (5), alismoxide (6) and guaia-1 (5), 6-diene (7), were isolated from gorgonian Muriceides collaris collected in South China Sea. Their structures were elucidated on the basis of extensive spectroscopic analysis [MS, IR, 1H NMR, 13C NMR (DEPT), HMQC, HMBC, NOESY] and by comparison of the spectral data with those of the literatures.

TLR4 mediates I/R injury involving endogenous ligands.Interaction of TLR4 with endogenous ligands provides a critical link between tissue damage and activation of the innate immune response.In the early phase of liver,kidney,heart,or lung I/R injury,endogenous ligands are secreted from several kinds of cells,they are recognized by TLR4.Interaction of TLR4 with endogenous ligands,such as HMGB1,seems to be the most important trigger of inflammation and initiates signaling cascades leading to inflammatory and immune responses.Blocking the interaction of TLR4 with endogenous ligands may be useful in clinical management of inflammation and cellular necrosis caused by ischemic insults.

OBJECTIVE: To investigate the effect of PNS on experimental atherosclerosis in rabbits, including the serum levels of TG, TC, LDL-C, level of MDA, activity of SOD and plaque area. METHODS: White Japanese rabbits were divided into normal control group, AS model group, low dose PNS group and high dose PNS group. Administration was for 12 consecutive weeks. The serum levels of TG, TC, LDL-C, MDA and activity of SOD were determined before experiment and at the end of the 12th week, respectively. RESULTS: The serum levels of TG, TC and LDL-C in AS model group were significantly higher than that in control group ( P

BACKGROUND: Lentivirus can infect divided and undivided cells. It remains uncertain whether the lentivirus can successful y infect primary ovarian granulosa cells. OBJECTIVE: To investigate infecting ratio and cel apoptosis of lentivirus carrying bcl-2 gene in primary human ovarian granulose cells cultured in vitro. METHODS: The lentiviral vector carrying bcl-2 gene was constructed using molecular biology, and packaged into lentivirus with high titer. The resulting recombinant lentivirus carrying bcl-2 genes were then used to infect primary human ovarian granulosa cells in vitro at different multiplicity of infection, 10, 50, 100, 200, and 400. Infection efficiency and cel proliferation were observed at 24, 48, 72, and 96 hours fol owing infection. Cel apoptosis was detected by flow cytometry, and bcl-2 gene transcription was assessed using reverse transcription PCR. RESULTS AND CONCLUSION: Primary human ovarian granulosa cells adhered at 24 hours, and exhibited polygon- or fusiform-shape and colony-like growth. When multiplicity of infection was 100, cel appearance and growth remained unchanged, and infection efficiency was high, which reached the peak up to 72 hours. Moreover, the positive rate was up to 60% in granulosa cells. Lentivirus carrying bcl-2 gene could increase expression of Bcl-2 protein and inhibit apoptosis of primary ovarian granulosa cells.

Objective To investigate the expressions of Toll-like receptor 4 (TLR4) and high mobility group box 1 (HMGB1) expression on distant tissue during the intestinal ischemia/reperfusion and the effects of ω-3 polyunsaturated fatty acids (ω-3 PUFAs) intervention in rats.Methods Forty-eight Sprague-Dawley male rats,weighing (281.50 ± 22.68) g,were randomly divided into three groups (n =16) after gastrostomy:normal diet (N) group,enteral nutrition (EN) group and EN plus ω-3 PUFAs (PUFA) group.Each group was further divided into lymph drainage (I/R + D) and non-drainage (I/R) sub-groups (n =8 each) according to whether treated with intestinal lymph drainage.All the rats were subjected to 60 min ischemia by clamping the superior mesenteric artery,followed by 120 min reperfusion,while the rats in the I/R + D subgroups were treated with intestinal lymph drainage for 180 min at the same time.Results The interleukin-6 level in lymph in N (I/R + D) group was significantly higher than in the EN (I/R + D) and PUFA (I/R + D) groups (PUFA vs EN vs N:(154.57 ±69.30) ng/L vs (97.58 ±40.34) ng/L vs (85.35 ±23.93) ng/L,P =0.021).Besides,the serum level of HMGB1 in PUFA (I/R + D) group was significantly lower compared to the other 5 groups [PUFA (I/R) vs EN (I/R) vs N (I/R) vs PUFA (I/R + D) vs EN (I/R + D) vs N (I/R + D):(2.95 ± 1.17) μg/L vs (3.86 ±0.99) μg/L vs (4.45 ± 1.73) μg/L vs (1.71 ±1.41) μg/Lvs (2.11±0.56) μg/Lvs (3.13 ±0.79) μg/L,P=0.000],and it also decreased in the PUFA (I/R) and EN (I/R) groups than the N (I/R) group (respectively,P ＜ 0.05).Furthermore,the serum endotoxin level in PUFA (I/R) group was significantly lower compared to the N (I/R) and EN (I/ R) groups[PUFA(I/R) vsPUFA (I/R+D) vsEN (I/R) vs N (I/R):(0.020±0.004) EU/mlvs (0.028 ±0.006) EU/ml vs (0.028 ±0.005) EU/ml vs (0.018 ±0.006) EU/ml,P=0.014].Together the serum tumor necrosis factor-α level in both PUFA (I/R) and PUFA (I/R + D) groups were significantly lower than theEN (I/R),N (I/R) and N (I/R+D) groups [PUFA (I/R+D) vs PUFA (I/R) vs EN (I/R) vsN (I/R) vs N (I/R+D):(12.03 ±6.57) ng/L vs (14.32 ±6.11) ng/Lvs (23.27 ±15.60)ng/L vs (27.42 ± 10.37) ng/L vs (26.87 ± 5.30) ng/L,P =0.013].The jejunum and ileum mucosa in all the I/R groups showed swelling and atrophy and appeared fragile,while the PUFA groups showed less yellow staining and injury than the other two groups (P ＜ 0.05,respectively).In addition,the expressions of TLR4 mRNA in jejunum,ileum,and liver in all the drainage groups were respectively lower than the corresponding non-drainage groups [jejunum:PUFA (I/R) vs EN (I/R) vs N (I/R) vs PUFA (I/R+D) vs EN (I/R+D) vsN (I/R+D):2.32±0.62vs3.08±1.29vs3.50±2.44vs 1.62±0.79vs 1.67±1.11 vs 1.94±0.81,P=0.025; ileum:PUFA (1/R) vsEN (1/R) vsN (1/R) vs PUFA (1/R+D) vsEN (1/R+D) vs N (1/R+D):2.67±1.08 vs 5.22 ± 3.96 vs 6.95 ±4.92 vs 1.70±0.68 vs 1.80±0.29 vs3.68±1.47,P=0.012; liver:PUFA (1/R)vsEN (1/R)vsN (1/R)vs PUFA (1/R+D)vsEN (1/R+D)vsN (1/R+D):5.67 ±1.94 vs 7.50 ±3.89 vs 7.18 ±4.55 vs 1.70 ±0.86 vs 3.90 ± 1.95 vs 4.12 ±2.11,P =0.001],which was consistent with the reduction of HMGB1 and the decrease of nuclear factor-κB activity in intestine,liver,and lung (P =0.000).Conclusions Lymph drainage and ω-3 PUFAs intervention can reduce the production of HMGB1 and inflammation factors,inhibit the expression of HMGB1 and TLR4 mRNA,and thus alleviate distant tissue injury caused by intestinal L/R.