Clinical evaluation of percutaneous endovascular radiofrequency ablation for portal vein tumor thrombus: experience in 120 patients.

BACKGROUND: Portal vein tumor thrombosis (PVTT) secondary to primary liver carcinoma (PLC) is commonly associated with poor prognosis and poses great challenge. This study was to evaluate the efficacy and safety of percutaneous endovascular radiofrequency ablation (RFA) in treatment of PVTT. METHODS: Consecutive patients who were performed endovascular RFA because of PVTT in single-institution in recent 8 years were retrospectively reviewed, compared with patients who underwent only sequential transcatheter arterial chemoembolization (TACE) during the contemporary period. Patency of portal vein, complications, and overall survival (OS) were investigated. RESULTS: One hundred and 20 patients who underwent endovascular RFA and 96 patients who underwent only sequential TACE were included. No severe complications happened in both groups. Except the higher rates of severe fever and moderate pain in the study group, no difference was found in the incidence of side effects and complications. The effective rate in the study group was (78.3%, 94/120) significantly higher than the comparison group (35.4%, 34/96). The median survival time and 1-3 years cumulative survival rates in the study group were 15.7 months and 42.5%, 21.7%, 2.5%, respectively, and 11.3 months, 21.9%, 9.4%, 0 correspondingly in the comparison group, without significant difference. Type of PVTT and Child-Pugh classification of liver function were independent risk factors, and OS was significantly improved by endovascular RFA and subsequent therapy. CONCLUSION: Endovascular RFA is technically safe and feasible for unresectable PLC and PVTT to improve the prognosis and quality of life.

Improving biliary stent patency for malignant obstructive jaundice using endobiliary radiofrequency ablation: experience in 150 patients.

BACKGROUND: Although self-expandable mental stents (SEMS) placement is the standard care for relieving obstructive jaundice caused by unresectable malignant biliary stricture, how to maintain stent potency remains an intractable problem. This study was to evaluate the efficacy and safety of endobiliary radiofrequency ablation (RFA) through percutaneous transhepatic cholangiography (PTC) pathway in treating such patients. METHODS: Consecutive patients who were performed endobiliary RFA as well as SEMS placement because of unresectable malignant obstructive jaundice in single institution in recent 8 years were retrospectively reviewed. As comparison, patients who underwent only percutaneous SEMS placement for unresectable malignant biliary stricture during the contemporary period were reviewed. Stent patency, complications, complications, and overall survival (OS) were investigated and analyzed. RESULTS: One hundred and fifty patients who underwent endobiliary RFA and 127 patients who underwent only stent placement were included in this study. In the study group of endobiliary RFA, 87 patients (58.0%) underwent ablation for 1 time, 49 (32.7%) for 2 times, and 14 (9.3%) for 3 times. Complications related to RFA as well as SEMS placement happened in 113 patients (75.3%), without severe complications that needed emergent surgery or interventional therapy. The median duration of stent patency after ablation was 11.2 month, and the median survival time was 12.3 month. As comparison, difference was found in the number of interventional procedures and stents placed, duration of initial stent patency, and the incidence of moderate bleeding and pain. In the study group, only the type of tumor that caused biliary obstruction (intrahepatic carcinoma vs. extrahepatic carcinoma) was a poor independent factor (P = 0.035) for recurrent biliary obstruction. Repeated interventional therapy and adoption of subsequent therapy were only independent factors for OS. CONCLUSIONS: Endobiliary RFA and SEMS placement is technically safe and feasible for unresectable malignant obstructive jaundice to improve the quality of life and prolong survival.

Modified Procedures for ALPPS Based on a Risk-Reduced Strategy: Paralleled Clinical Evaluation at Multiple Institutions.

PURPOSE: We compared the clinical outcomes of modified procedures for associating liver partition and portal vein ligation for staged hepatectomy (ALPPS) based on a risk-reduced strategy with those of classic ALPPS procedures in treating large liver carcinoma. MATERIALS AND METHODS: Short-term outcomes, increases in future liver remnant (FLR) and functional FLR (FFLR), and overall survival (OS) were compared between 45 consecutive patients treated with modified ALPPS procedures and 34 patients treated with classic ALPPS procedures. RESULTS: Clinical outcomes after the 1st-stage operation markedly improved with the modified procedures. Although the proportions of liver cirrhosis and hepatocellular carcinoma were higher in the modified group, the mortality and incidence of severe complications did not increase. FLR and FFLR hypertrophy at 1 week after the 1st-stage operation were similar in both groups; however, kinetic growth rates in the modified group were lower. OS rates were similar. CONCLUSION: Modified ALPPS procedures could be safely applied to provide long-term survival for patients with liver cirrhosis without sufficient FLR.

Short-Term and Long-Term Outcomes of Laparoscopic Versus Open Surgery for Low Rectal Cancer.

AIM: To compare the short-term and long-term outcomes of laparoscopic versus open surgery for low rectal cancer. METHODS: Patients with low rectal cancer who underwent laparoscopic or open surgery at our department from January 2009 to December 2013 were enrolled in this retrospective study. The primary end points were 3-year local recurrence and overall and disease-free survival (DFS) rates. Secondary end points were intraoperative and postoperative outcomes. RESULTS: Laparoscopic group had longer operative time (165.0 versus 140.0, P < .001), less blood loss (20.0 versus 40.0, P < .001), shorter length of incision (5.0 versus 18.0, P < .001), and more lymph node harvested (11.0 versus 9.0, P = .002). However, time to first flatus (P = .941), postoperative hospital stay (P = .095), postoperative complications (P = .155), and 30-day mortality (P = .683) was similar between two groups. With the median follow-up period of 65 months, the 3-year local recurrence rate was 4.3% in laparoscopic group and 7.5% in open group (P = .077); the 3-year overall and DFS rates were similar in two groups (85.9% versus 88.8%, P = .229 and 76.9% versus 79.2%, P = .448, respectively); and the overall and DFS curves were comparable between two groups (hazard ratio [HR] = 0.858, 95% confidence intervals [CI] 0.709-1.037, P = .112 and HR = 1.076, 95% CI 0.834-1.389, P = .275, respectively). CONCLUSIONS: Laparoscopic surgery is safe and has equivalent long-term oncologic outcomes for low rectal cancer when compared to open surgery. Furthermore, large-scale, prospective randomized clinical trials are needed to confirm the present findings.

Study on the supramolecular systems of 5-(2-hydroxy phenyl)-10,15,20-tris (4-methoxy phenyl) porphyrin with cyclodextrins.

In neutral phosphate buffer solutions of pH 7.4, the inclusive complexation of 5-(2-hydroxy phenyl)-10,15,20-tris(4-methoxy phenyl) porphyrin (o-HTPP) with alpha-cyclodextrin (alpha-CD), beta-CD, heptakis (2,3,6-tri-O-methyl)-beta-CD (TM-beta-CD), SBE-beta-CD, HP-beta-CD and gamma-CD has been examined by means of UV-vis and fluorescence spectroscopy. The formation of inclusion complexes has been confirmed on the base of changes of spectroscopy properties. The o-HTPP forms 1:2 inclusion complexes with TM-beta-CD and 1:1 inclusion complexes with the other five cyclodextrins. The formation constants (K) of o-HTPP for the formation of the inclusion complexes have been estimated from the absorbance and fluorescence intensity changes in neutral phosphate buffer solutions. The K value (2.89x10(7)), which is the formation constant for the formation of the 1:2 inclusion supramolecular, is nearly 10(4) times than those of the 1:1 inclusion complexes. Compared to the other five cyclodextrins, the strongest inclusion ability of TM-beta-CD can be explained that the hydrogen bond plays significant role in the inclusion process. UV-vis experiments also showed that the cavity of TM-beta-CD causes the transform of the state of o-HTPP. In addition (1)H NMR data and 2D-ROSEY NMR spectra support the inclusion conformation of the o-HTPP-CD supramolecular system, indicating the interaction mechanism of inclusion processes.

[Clinical outcome of patients with follicular development retardation by prolonged duration of gonadotropin administration for in vitro fertilization].

OBJECTIVE: To investigate prolonged use of gonadotropin (Gn) stimulation for the patients with follicular development retardation during controlled hyperstimulation (COH) for in vitro fertilization (IVF) cycles. METHODS: The inclusion criteria to categorize the good responder were that duration of Gn stimulation was < or = 15 days, total Gn dose used was < or = 3300 IU and total oocytes retrieved were > or = 4. The inclusion criteria for prolonged duration of Gn stimulation were that duration of stimulation was > or = 16 days. There were 69 oocyte retrieval cycles and 66 transfer cycles in prolonged stimulation group (group 1) and 483 oocyte retrieval cycles and 464 transfer cycles in good ovarian response group (group 2). The clinical characteristics and outcomes of two groups were analyzed. RESULTS: Clinical 5 pregnancy rate, implantation rate and delivery rate were 45.5% (30/66) versus 51.7% (240/464), 30.5% (385/1262) versus 28.0% (46/164) and 37.9% (25/66) versus 39.4% (183/464), respectively, in groups 1 and 2. Duration of Gn stimulation and dose were (20.8 +/- 4.2) and (10.3 +/- 1.8) days, (3090 +/- 1140) and (2302 +/- 862) IU in groups 1 and 2, respectively. There was no statistical difference in patient's age, basal follicular stimulating hormone (FSH), clinical pregnancy rate, implantation rate, early miscarriage rate, ovarian hyperstimulation syndrome (OHSS) rate, delivery rate between two groups (P > 0.05). There were more polycystic ovary (PCO) and (or) polycystic ovarian syndrome (PCOS) patients, more basal antral follicles, longer duration of Gn stimulation (range 16 - 33 days), higher Gn dose, lower serum peak estradiol (E(2)) level, fewer oocytes, fewer embryos transferred, in group 1 compared with group 2 (P < 0.01). CONCLUSIONS: These results demonstrate that prolonged Gn stimulation more than 16 days is a valuable alternative before cancellation of the IVF cycles for follicular development retardation during COH. Good clinical outcome can be achieved including pregnancy rate, implantation rate and delivery rate.

Construction of influenza virus siRNA expression vectors and their inhibitory effects on multiplication of influenza virus.

Three plasmid constructs were prepared that express small interfering RNAs (siRNAs) targeted to sequences encoding the ribonucleoprotein member, nucleoprotein (NP) and/or PA, of influenza virus genome. The antiviral properties of siRNAs against the H5N1 strain of influenza virus were studied by evaluating their capacity to silence expression of target genes as well as their effect on influenza virus-induced apoptosis in Madin-Darby canine kidney cells, chicken embryo fibroblast cells, and embryonated chicken eggs in a transient replication model. The results demonstrated that all three siRNAs expressing plasmids efficiently transcribed the short hairpin RNAs and inhibited expression of the NP or PA proteins measured by northern blot and western blot analyses, respectively, in the transfected cells. We also found that the integrated siRNA expression plasmid pEGFP/NP+PA, which we constructed for the first time to synchronously target NP and PA segments of the influenza virus genome, could more efficiently inhibit synthesis of influenza virus detected by cytopathogenic effects, hemagglutinin, and plaque-forming unit assays in the transfected cells. Furthermore, the integrated siRNA expression plasmid pEGFP/NP+PA could remarkably interrupt the cellular apoptotic course caused by influenza virus, which protected infected cells from apoptotic damage. In contrast, a control siRNA expression plasmid, pEGFP/HK, could neither inhibit the protein expression and production of influenza virus nor interrupt the cell apoptotic course mediated by influenza virus. These results demonstrate that RNA interference (RNAi) can be used to inhibit protein expression and replication of influenza virus and that RNAi treatment holds potential as a new approach to prevent avian influenza.

Expression of imprinted genes related to Beckwith-Wiedemann syndrome in human oocytes and preimplantation embryos.

OBJECTIVE: To investigate the expression of imprinted genes related to Beckwith-Wiedemann syndrome (BWS) in human oocytes and preimplantation embryos for understanding the relationship between assisted reproductive technology (ART) and BWS. METHODS: Using nested reverse transcription-PCR to analyze the expression of P57KIP2, LIT1, TSSC3 in human oocytes and preimplantation embryos. RESULTS: Transcripts of P57KIP2 were detected in human oocytes and at all stages of preimplantation embryos. LIT1 was expressed only in stages of 8-cell and blastocyst. Transcripts of TSSC3 could not be detected in human oocytes and preimplantation embryos. CONCLUSION: Transcripts of P57KIP2 and LIT1, imprinted genes related to BWS, were detected in human preimplantation development; ART might affect the epigenetics of imprinted genes in early embryogenesis.

[Expression of imprinted gene Grb10 in human oocytes and preimplantation embryos].

OBJECTIVE: To investigate the mRNA expression of imprinted gene Grb10, a candidate gene for Silver-Russell syndrome (SRS), in human oocytes and preimplantation embryos for understanding the role of Grb10 in embryogenesis and assisted reproductive technology (ART). METHODS: Nested reverse transcriptase (RT)-PCR was used to analyze Grb10 gene expression in human oocytes and preimplantation embryos. RESULTS: The transcripts of Grb10 were detected in human oocytes and preimplantation embryos at all stages except for the 2-cell stage. CONCLUSION: Grb10 gene is expressed in preimplantation embryos with close association with embryogenesis.

[Mitochondria transfer from self-granular cells to improve embryos' quality].

OBJECTIVE: To improve embryos' quality and pregnancy rate with the method of self-mitochondria transfer. METHODS: All the 18 cases of women with repeated in vitro fertilization-embryo transfer (IVF-ET)-treatment failure or older than 37 years were treated with the long gonadotropin releasing hormone-agonist (GnRH-a) regimens. The oocytes were divided into two groups: intracytoplasmic sperm injection (ICSI) group and mitochondria transfer group if the number of the individual patients' oocytes was more than four. In mitochondria transfer group, mitochondria were prepared by different centrifugation after the granulosa cells were trimmed from the oocytes or collected from follicular fluid and homogenated. Mitochondria and immobilized sperm were injected into oocytes by micromanipulation. The fertilization rate and embryos' quality were compared. RESULTS: The fertilization rate was 74.4% in mitochondria transfer group, and 76.8% in ICSI group, with no statistical difference (P > 0.05). The good quality embryo rate in mitochondria transfer group was 59.4%, and it was 34.9% in ICSI group. The difference was significant (P < 0.05). There were 7 clinical pregnancies in the 18 cases. CONCLUSION: Self-mitochondria transfer can improve embryos' quality and pregnancy rate without influence on fertilization rate.

[Transforming growth factor-beta1, estradiol, progesterone and lutropin levels in follicular fluid after ovarian stimulation].

OBJECTIVE: To investigate the role of transforming growth factor-beta1 (TGFbeta1) and the sex hormones in the follicular fluid (FF) on the day of ovum pick-up (OPU) during controlled ovarian hyperstimulation (COH) cycles. METHODS: FF and the oocytes were obtained from the follicles of 90 women undergoing ovulation stimulation in vitro fertilization (IVF) treatment. TGFbeta1, estradiol, progesterone, and lutropin concentrations in the FF samples collected during transvaginal oocyte retrieval were measured using enzyme-linked immunosorbent assay (ELISA). The maturity and fertilization of the oocytes were observed, and ultrasonography performed to confirm clinical pregnancies 4 weeks after the embryo transfer. RESULTS: In the FF containing mature oocytes, progesterone and lutropin concentrations were significantly higher than those in the FF with immature oocytes. The mean concentrations of TGF beta1, progesterone and lutropin in the FF from fertilized subjects were obviously higher than those in the FF from non-fertilized subjects (P < 0.05), and in subjects with pregnancy, higher mean concentrations of TGFbeta1 and lutropin were detected as compared with the concentrations measured from non-pregnancy subjects (3 631.4+/-1 426.3 pg/ml and 0.74+/-0.25 mIU/ml vs 2 189.2+/-1 180.4 pg/ml and 0.52+/-0.29 mIU/ml respectively, P < 0.05). Estradiol concentrations in the FF seemed to undergo no obvious changes during the whole procedure, and evinced no significant differences between the groups. CONCLUSION: Higher lutropin and progesterone concentrations in the FF on the day of OPU may promote oocyte maturation, while TGFbeta1 and lutropin levels appear to be associated with the maturation and fertilization of the oocytes, and may be indicative of the IVF outcome.

[First twins born in Mainland China by autologous granular cell mitochondria transfer].

A 37-year-old woman with the history of twice spontaneous abortion, who received unfruitful in vitro fertilization and embryo transfer (IVF-ET) for two cycles due to poor embryo quality, underwent the third cycle for autologous granular cell mitochondria transfer of 5 matured oocytes and intracytoplasmic sperm injection (ICSI) in another 4 oocytes. Better embryo quality was resulted by the former technique than by the latter, despite that fertilization was achieved in all the 4 oocytes with the latter operation. Four of the 5 oocytes with mitochondria transfer were fertilized and developed into normal embryos, 3 of which were selected for intrauterine transplantation. Clinical pregnancy with triplets was subsequently obtained, but the development of one fetus accidentally stopped at the fifth week of pregnancy. The woman received five times of immunotherapy with her husband's leukocytes before and after the pregnancy. At the 30 th week of pregnancy, the women was admitted for severe pregnancy-induced hypertension syndrome that failed to respond to a two-week treatment course. Cesarean section was subsequently performed and two normal babies, one boy and one girl, were born on August 6th, 2003, who were taken home after care in neonate wards. This is the first report of the birth of babies born by autologous granular cell mitochondria transfer in Mainland China.

[The first polar body of human oocytes does not help predict the precise location of the spindle].

OBJECTIVE: To observe the precise position of the metaphase II spindle for facilitating clinical practice of assisted reproduction. METHODS: LC-PolScope imaging system was used for observing the spindles in human metaphase II oocytes matured in vitro or in vivo, and the angle between the center of the oocyte, the spindle and the first polar body was measured and compared between in vitro and in vivo matured oocytes. RESULTS: In vitro and in vivo matured oocytes were significantly different in the angle between the spindle and the first polar body (P=0.006). CONCLUSION: The deviation of the spindle from the polar body in in vitro matured oocyte was significantly smaller than that in in vivo matured oocyte, and the first polar body does not help predict the precise location of the metaphase II spindle. The imaging system is safe and effective for observing the spindles.

Pregnancy in a 46-year-old woman after autologous granular cell mitochondria transfer.

Growth of 4 follicles was achieved in a 46-year-old infertile woman and 2 mature oocytes were subsequently obtained after pituitary desensitization with triptorelin (1.875 mg) and hyperstimulation with recombinant human FSH (Gonal-F) and urinary human menopausal gonadotropin (HMG). The granular cells were collected from the cumulus and follicle fluid of the patient with Percol gradient centrifugation. The cells (about 1 x 10(6) /ml) were homogenized for 6 times with cold glass homogenizer, the resultant crude homogenate centrifuged for 20 min at 2000 x g. The supernatant was again centrifuged for 20 min at 9800 x g, after which the pellets of the mitochondria were resuspended in 1 microl modified human tubal fluid (mHTF) medium and maintained at 4 degrees Celsius. A microinjection needle with the inner diameter of approximately 7 microm was used to aspirate the mitochondrion suspension for about 100 microm in length on the scale of the needle for delivering the mitochondria, along with the immobilized sperms, into a single MII oocyte. About 3000 mitochondria were transferred into each oocyte. The two oocytes were both fertilized, but one had 3 pronucleus, and the normal fertilized oocyte developed into 8-blastomere embryo 3 d after its transfer into the patient's uterus. The urine test confirmed clinical pregnancy in the woman 15 d later, and 30 d after the transfer, the fetal body and heart beat could be observed by ultrasound, but unfortunately, spontaneous abortion occurred at the ninth week. According to our knowledge, this woman is the eldest person in mainland China to receive assisted reproduction operations that successfully got clinical pregnancy with her own oocyte.

[Expression of uncoupling protein 2 in periimplantation endometrium and its relationship with infertility].

OBJECTIVE: To study the expression of uncoupling protein 2 (UCP2) mRNA and protein in the endometrium and its changes during periimplantation period in relation to infertility. METHODS: In situ hybridation and Western blotting were used to detect 21 endometrium specimens from normal cycling women during different menstrual phases, 22 secretory endometrium specimens from infertile women and 7 decidual and chorionic villus specimens. RESULTS: UCP2 mRNA and protein were detected in the endometrium and decidua besides, the trophoblast, and UCP2 level in the endometrium increased during pregnancy. Compared with normal women, women with hydrosalpinges had significantly stronger endometrial UCP2 expression in the mid-luteal stage. CONCLUSIONS: UCP2 down-regulates reactive oxygen species and plays a role in the regulation of inflammatory events to prepare for embryo implantation. Strong endometrial UCP2 expression might be an important reason that hydrosalpinges affect embryo implantation.

[Allogeneic leukocyte immunization combined with IVF-ET for treatment of infertility induced by recurrent spontaneous abortion].

OBJECTIVE: To investigate the effect of allogeneic leukocyte immunization combined with in vitro fertilization-embryo transfer (IVF-ET) for treatment of infertility induced by habitual abortion. METHODS: Allogeneic leukocyte immunization was performed in 9 patients with infertility induced by habitual abortion, with another 9 patients undergoing IVF-ET without habitual abortion as the control group. All the patients were treated with long GnRH-a protocols. The infertility patients with recurrent spontaneous abortion history were immunized with lymphocytes from the husband for before IVF-ET and after clinical pregnancy. RESULTS: The fertilization rates of the immunotherapy group and control group were 81.3% and 82.2%, respectively, showing no significant difference (P>0.05). Five patients in each group had clinical pregnancy, and a twin pregnancy occurred in the control group. The embryo implantation rates were also comparable between the two groups (22.7% vs 28.6%, P>0.05). All the fetuses resulted from IVF-ET developed normally and were healthily delivered. CONCLUSION: Allogeneic leukocyte immunotherapy along with IVF-ET is effective for treatment of infertility resulting from recurrent spontaneous abortion.

[Neonatal outcomes of pregnancy resulting from in vitro fertilization and embryo transfer: analysis of 1274 cases].

OBJECTIVE: To evaluate the neonatal outcomes of pregnancies resulting from in vitro fertilization and embryo transfer (IVF-ET) in relation to the occurrence of congenital malformations of the neonates. METHODS: A total of 1274 infants born after IVF-ET were reviewed. The neonatal outcome was evaluated based on gestational weeks, body weight, congenital malformations, manner of spermatization, maternal age and multiple gestation. RESULTS: IVF-ET resulted in 930 deliveries, giving birth to a total of 1274 newborns. Spontaneous delivery occurred in 115 cases (12.37%), with preterm birth in 224 cases (24.09%). Among these newborns, 363 (28.49%) had very low born weight (VLBW), 13 (1.02%) had congenital malformations, and neonatal mortality occurred in 15 cases (1.18%). CONCLUSION: IVF increases the risks of twin pregnancies, preterm birth and VLBW, but does not increase the rate of congenital malformations and neonatal mortality. Intracytoplasmic sperm injection (ICSI) is at higher risk of congenital malformations. Maternal age and twin pregnancies are not associated with congenital malformations. IVF can be safe for treatment of infertility.

[Inducement effect of recombinant human TNF-alpha on apoptosis of breast cancer cell line ZR75-1 and Its mechanism].

BACKGROUND & OBJECTIVE: Tumor necrosis factor alpha (TNF-alpha) is an important agent for tumor biotherapy because of its strong antitumor activity, but its clinical application is limited because of its severe fatal systemic toxicity. A new recombinant human (rh) mutein, mutant type (mt) 471rhTNF-alpha, was produced to decrease toxicity while keep antitumor activity of wild type (wt) TNF-alpha. This study was to compare the antitumor activities of mt-471rhTNF-alpha and wt-rhTNF-alpha, and analyze the mechanism of tumor cell apoptosis induced by mt-471rhTNF-alpha. METHODS: Breast cancer cell line ZR75-1 was treated with 100 microg/L of mt-471rhTNF-alpha or wt-rhTNF-alpha for 12 h. Cell apoptosis was analyzed with 2% agarose gel electrophoresis, and cell cycle distribution was analyzed by flow cytometry. The activation of nuclear factor-kappaB (NF-kappaB) p65 in ZR75-1 cells treated with 10 microg/L, 50 microg/L, and 100 microg/L of mt-471rhTNF-alpha or wt-rhTNF-alpha for 4 h were detected using Trans AMTM NF-kappaB p65 kit based on ELISA. RESULTS: ZR75-1 cells treated with mt-471rhTNF-alpha had typical ladders of DNA fragmentation, whereas ZR75-1 cells treated with wt-rhTNF-alpha only had weak ladders; the apoptosis rate of ZR75-1 cells was significantly higher in mt-471rhTNF-alpha group than in wt-rhTNF-alpha group [(44.6+/-3.6)% vs. (24.7+/-2.7)%, P<0.05]. The proportion of G1 phase cells was significantly higher in mt-471rhTNF-alpha group than in control group and wt-rhTNF-alpha group [(66.6+/-4.2)% vs. (34.8+/-3.1)% and (46.2+/-3.8)%, P<0.05]; the proportions of S phase and G2 phase cells were significantly lower in mt-471rhTNF-alpha group than in control group and wt-rhTNF-alpha group [(33.2+/-2.9)% vs. (50.1+/-3.8)% and (45.7+/-2.9)%, P<0.05; (0.2+/-0.0)% vs. (15.1+/-2.2)% and (8.1+/-3.1)%, P<0.05]. When treated with 50 microg/L rhTNF-alpha for 4 h, NF-kappaB activation was significantly higher in wt-rhTNF-alpha group than in mt-471rhTNF-alpha group (3.3+/-0.1 vs. 1.5+/-0.2, P<0.05 ). CONCLUSIONS: The antitumor ability of mt-471rhTNF-alpha is more stronger than that of wt-rhTNF-alpha. The explanation for the enhanced apoptosis-inducible ability of mt-471rhTNF-alpha could be that NF-kappaB activation is inhibited in tumor cells treated with mt-471rhTNF-alpha.

[Study on the preliminary purification and bioactivity of recombinant human TNF-alpha mutein 471].

AIM: To purify preliminary recombinant human TNF-alpha mutein 471 and detect its bioactivity on the basis of the TNF-alpha mutein 471 expressed in prokaryotic express system. METHODS: The expression of recombinant human TNF-alpha mutein 471 in engineering bacteria strains E.coil was induced under the condition of optimal fermentation and expression. After cultured E.coil cells were collected and broken by using an ultrasonic disintegrator, the TNF-alpha mutein 471 existed in the form of inclusion body was extracted and purified, and then the effects of denaturation and protein concentration on protein folding were examined. The bioactivities of wild type TNF-alpha and the TNF-alpha mutein 471 were detected by MTT colorimetry. RESULTS: The TNF-alpha mutein 471 was folded and polymerized successfully to from a trimer with bioactivity under the condition of proper denaturation and renaturation. The cytotoxic activity of the TNF-alpha mutein 471 to the L929 cells was 15 times as much as wild type TNF-alpha. CONCLUSION: The TNF-alpha mutein 471 expressed in prokaryotic expression system possesses significantly bioactivity after renaturation, which lays the foundation for further animal experiment and clinical experimental researches.

[Study on immunoregulatory functions of E.coli heat-labil enterotoxin B subunit].

AIM: To explore the immunoregulatory functions of Escherichia coli heat-labil enterotoxin B subunit(LTB) and its possible mechanism. METHODS: The rLTB in engineering bacteria VSP60 was purified by Sephacryl S-100 gel filtration chromatography. BALB/c mice were immunized nasally with hen egg lysozyme(HEL) alone or in combination with various doses of LTB. After three times of immunization, specific anti-HEL IgG and IgA levels in serum and small intestinal secretory fluid were determined by ELISA. The effects of LTB on alloantigen- and ConA- mediated lymphocyte proliferation reactions were measured by (3)H-TdR incorporation assay. RESULTS: Very weak serum anti-HEL IgG response and no IgA response were detected in mice immunized with HEL alone. However, levels of both serum anti-HEL IgG, IgA and intestinal secretory fluid IgA in mice immunized with various doses of HEL+LTB were higher than those in mice immunized with HEL alone(P<0.001). In-vitro, LTB could obviously inhibit the lymphocyte proliferation induced by mitogen and alloantigen. CONCLUSIONS: The effects of LTB protein on immnune system may be bi-directional. LTB is not only a powerful mucocal immunoadjuvant in-vivo, but also a potent immunosuppressive agent In-vitro.