BACKGROUND:Xenogeneic bone structure and biological characteristics are similar to human bone tissue, and the xenogeneic bone has a decreased antigenicity after physicochemical treatment, with a natural porous structure and rich source, and can be kept for a long time, which is considered to be an effective way to solve the shortage of the autogenous bone and al ograft bone.
OBJECTIVE:To compare the physical and chemical properties of xenogenic bone materials prepared by two different methods.
METHODS:Sheep cancellous bone treated with chemical method was placed into the muffle furnace at 1 000 ℃for 2 hours to prepare calcined bone. Another cancellous bone was placed into an 80 ℃ refrigerator for 4 weeks and then placed into a vacuum instrument to prepare freeze-dried bone. Cancellous bone rinsed with ultra-pure water served as controls.
RESULTS AND CONCLUSION: Three groups of samples retained three-dimensional porous structure which similar with human bone tissue under microscopic observation. The framework was stil intact, with a smal pore of 55-650μm and high porosity of 65%-80%. For the calcined bone, the toughness was decreased and the brittleness increased significantly, but the freeze-dried bone had a little changes in the mechanical properties. Through diffraction analysis, hydroxyapatite was the main composition of the three groups. However, there was a smal amount ofβ-tricalcium phosphate in the calcined bone. Spectrum analysis confirmed that calcium and phosphorus content in these three groups were al close to the human body. The results suggest the cancellous bone treated with these two methods is similar to human bone structure, and the major elements are close to the body. In addition, the cancellous bone after processing has enough smal pore and higher porosity. However, calcination process has a more influence on the mechanical property of scaffold materials, and the freeze-dried bone has a little change but the antigen cannot be completely removed that can reach the basic requirements.

ObjectiveTo investigate whether CD4+ CD25+ CD127dim/- regulatory T lymphocytes (Treg) can induce the proliferation of hepatic stellate cells (HSC) and expression of fibrosis-related factors on HSC in vitro and further to explore the mechanism of Treg inducing fibrogenesis. MethodsHSC LX-2 cells were subcultured.CD4+ CD25+ CD127dim/- cells were purified using magnetic cell separation. The HSC were co-cultured with Treg by direct contact or by Transwell system in vitro. The HSC cultured alone was used as control. Cell proliferation was measured by CCK-8 assay.The expression of transforming growth factor (TGF)-β1 was detected by enzyme-linked inmunosorbent assay (ELISA), and the expressions of hyaluronic acid (HA) and precollagen Ⅲ (PC Ⅲ ) were detected by radio immunoassay (RIA). The data were analyzed by LSD-t test. ResultsHSC proliferation was strongest when Treg∶ HSC= 1.5∶ 1. The absorbance in direct contact co-culture group and Transwell system co-culture group was (0. 713±0. 032) cpm and (0. 735±0. 028) cpm, respectively, both of which were higher than that in control group [(0. 677 ± 0. 029) cpm](t = 5. 4003 and 8. 7878,respectively; both P＜0. 01). The concentrations of TGF-β1 in the supernatant were (781. 59 ±76.45) pg/mL and (813. 53±60. 62) pg/mL, respectively in direct contact co-culture group and Transwell system co-culture group, which were significantly higher than that in control group [(722.51±59. 66) pg/mL](t = 4.0014 and 6. 1653, respectively; both P＜0.01).The concentrations of HA were (433. 575±27.90) ng/mL and (445.40±23.73) ng/mL, respectively in direct contact co-culture group and Transwell system co-culture group, which were higher compared to that in control group [-(415. 83±19.44) ng/mL](t =3. 3124 and 5. 5231, respectively; both P＜0.01). Likewise, the concentrations of PCⅢ were (21. 93± 1.71) and (23. 125± 1.87) ng/mL in direct contact group and Transwell group, respectively compared to (20. 10± 1.49) ng/mL in control group (t = 4. 8082 and 7. 9436, respectively; both P ＜ 0.01).Furthermore, the absorbance,concentrations of TGF-β1, HA and PC Ⅲ in Transwell co-culture group were all higher compared to direct contact group (t = 3. 3875, 2.1639, 2. 2107 and 3.1354, respectively; all P＜0. 05).ConclusionsThe cell proliferation and the expressions of fibrosis-related factors in HSC increase greatly after co-cultured with CD4+ CD25+ CD127dim/-Treg. Therefore, Treg may play an important role in inducing liver fibrogenesis.

Objective To investigate the roles of five scoring systems including model for endstage liver disease (MELD), Child-Turcotte-Pugh (CTP), Mayo, MESO and MELD-Na scoring systems, in predicting the prognosis of patients with chronic severe hepatitis. Methods The clinical data of 213 patients with chronic severe hepatitis were retrospectively studied. The five scoring systems were applied respectively to evaluate the scores in survival group and death group. The capability of these five scoring systems to predict the prognosis of severe hepatitis were compared by the receiver operating characteristic (ROC) curve, area under curve (AUC) and cut-off value.Measurement data were compared by group t test. The comparisons of AUC among scoring systems were done using MEDCLAC software. Results The scores of death group evaluated by MELD, CTP,Mayo, MESO or MELD-Na scoring systems (30.6 ± 9.5, 11.3 ± 1.5, 10.4 ± 1.3, 2.3 ± 0.8 and 39.0 ± 11.8, respectively) were consistently higher than those of survival group (21.1± 6.8, 10.6 ±1.6, 9.0±1.5, 1.6±0.5 and 22.6±8.2, respectively) (P＜0.01). The values of AUC of these five systems were 0.810, 0.623, 0.749, 0.829 and 0.885, respectively. The Youden's indexes of these five systems were 0.507, 0.175, 0.389, 0.528 and 0.650, respectively. Conclusions The CTP scoring systems can not predict the prognosis of chronic severe hepatitis very well. The Mayo scoring systems can partially predict the prognosis. On the contrary, MELD, MESO and MELD-Na systems can successfully predict the disease prognosis, and the score of MELD-Na system shows the best correlation with the prognosis.

Objective To investigate the practical efficacy and safety of ultrasound with microbubbles mediated rAAV2-EGFP to retina of rat after intravitreal and subretinal injection. Methods Gene transfer was examined by rAAV2-EGFP intravitreal and subretinal injection into the Wistar rats with or without microbubbles. The eyes were exposed to US (1 MHz,2 W/cm2, duration 5 minutes,duty cycle 50%,pulse recurrent frequency 100 Hz). The onset of EGFP gene expression, lightness of fluorescence, area of fluorescence and its distribution in the fundus in vivo via fluorescence stereosocope were investigated on the 4th,7th, 35th,49th and 120th day respectively. The value of gene transfer was quantified through the EGFP fluorescence quantitative methods by Axiovision 3. 1 software. HE staining was used to observe tissue damage. Results There was no fluorescence observed by fluorescence stereosocope after intravitreal injection after two-month study. After subretinal injection, ultrasound-targeted microbubbles destruction (UTMD) strongly increased gene transfer efficiency. UTMD used in the experiment did no harm to the rat retina structure. Conclusions UTMD could not enhance rAAV2-EGFP transfecion efficiency to rat retina after intravitreal injection but the transduction could be enhanced significantly after subretinal injection.

OBJECTIVETo observe the effects of adipose-derived mesenchymal stem cells (ADSCs) with continous over-expression of glial cell line-derived neurotrophic factor (GDNF) on the motor function recovery and nerve regeneration of sciatic nerve of rats after electrical injury.

METHODSFive SD rats were collected to prepare ADSCs with over-expression of GDNF. One hundred and fifty SD rats were divided into normal control group (N), GDNF-ADSCs group (GA), ADSCs group (A), GDNF group (G), and physiological saline group (P) according to the random number table, with 30 rats in each group. Rats in group N were routinely fed without treatment, and rats in the other 4 groups were inflicted with electrical injury on sciatic nerve of thigh of the right hind leg. Rats in groups GA, A, G, and P were respectively injected with 100 µL suspension of ADSCs with over-expression of GDNF (1 x 10(7) cells per mL), 100 [µL ADSCs suspension (1 x 10(7) cells per mL), 100 µL GDNF solution (100 mg/L) , and 100 µL physiological saline to the surface of the injured nerves immediately after injury. Six rats of each group were collected for measuring hind limb stride from post injury week (PIW) 1 to 8, and morphology of the sciatic nerves was observed in PIW 8. In PIW 4, the protein expression of GDNF of sciatic nerves of the rest rats in each group was determined with Western blotting. Data were processed with one-way analysis of variance, analysis of variance of repeated measurement, and SNK test.

RESULTSCompared with that of group N, the hind limb stride values in groups GA, A, G, and P were significantly lower at each time point (with P values below 0.05). Compared with those of group P, the hind limb stride values in group GA from PIW 3 to 8, in group A in PIW 3, 5, and 7, and in group G in PIW 3, 5, 7, and 8 were significantly longer (with P values below 0.05). The hind limb stride values in group GA from PIW 4 to 8 were respectively (10.83 ± 0.97), (13.25 ± 1.40), (12.86 ± 1.42), (14.06 ± 1.50), and (15.09 ± 1.17) cm, which were significantly longer than those in group A [(8.90 ± 0.82), (9.03 ± 0.57), (9.27 ± 0.36), (9.86 ± 0.36), and (9.52 ± 0.58) cm] and group G [(8.87 ± 0.69), (8.51 ± 1.18), (9.34 ± 0.87), (9.76 ± 0.67), and (9.50 ± 1.22) cm], with P values below 0.05. Compared with that of group N, the number of myelinated nerve fibers of sciatic nerves was obviously decreased in group P but obviously increased in groups GA, A, and G; the diameter of axons was obviously shorter, and the myelin thickness was obviously increased in groups GA, A, G, and P in PIW 8 (with P values below 0.05). The number of myelinated nerve fibers in group GA was 31.2 ± 0.8, which was significantly higher than that in group A (23.7 ± 2.7), group G (22.3 ± 2.7), or group P (9.3 ± 2.8), with P values below 0.05. The diameter values of axons among groups P, A, G, and GA were similar (with P values above 0.05). The myelin thickness of rats in group GA was (3.41 ± 0.34) µm, which was significantly thicker than that in group A [(2.64 ± 0.37) µm] or group G [(2.41 ± 0.34) µm], with P values below 0.05. In PIW 4, the protein expression of GDNF of sciatic nerves was significantly higher in groups P, A, G, and GA than in group N (with P values below 0.05), and the protein expression of GDNF in group GA was significantly higher than that in group P, A, or G (with P values below 0.05).

CONCLUSIONSADSCs over-expressing GDNF protein can obviously promote the motor function recovery and nerve regeneration of sciatic nerve of rats after electrical injury.

Adipose Tissue ; Animals ; Electrophysiology ; Glial Cell Line-Derived Neurotrophic Factor ; genetics ; metabolism ; Mesenchymal Stem Cell Transplantation ; methods ; Mesenchymal Stromal Cells ; metabolism ; Nerve Crush ; Nerve Regeneration ; physiology ; Rats ; Rats, Sprague-Dawley ; Sciatic Nerve ; pathology ; physiology

Objective To investigate the transfection efficiency of combination of ultrasound (US) microbubbles (MBs) mediated liposome small interference RNA (siRNA) to human and rat retinal pigment epithelium (RPE) cells. Methods Human and rat RPE cells and siRNA were incubated in 24-well plates (2×10~5/well and 1×10~5/well, respectively). The cells were devided into 5 groups: siRNA+US, siRNA+MBs+US, siRNA+L (liposome), siRNA+L+US, siRNA+L+US+MBs. After 12 h, transfection efficiency was observed with fluorescence microscopy and flow cytometry. Results US or ultrasound targeted microbubbles destruction without liposome-mediated could not promote siRNA transfection efficiency to human and rat RPE cells. The transfection efficiency of human and rat RPE cells significantly decreased in the siRNA+L+US+MBs group, but increased in siRNA+L+US group. Conclusion Ultrasonic irradiation can promote lipid-mediated siRNA transfected human RPE cells.

Objective: To investigate the effect of intracoronary administration of nicorandil prior to primary percutaneous coronary intervention (PPCI) on myocardial perfusion and short-term clinical outcomes in patients with ST-segment elevation myocardial infarction (STEMI). Methods: A total of 158 patients with STEMI undergoing PPCI from January 2014 to December 2015 in Fuzhou General Hospital were enrolled consecutively in this prospective controlled randomized trial. Patients were assigned into three groups with random number table: the nicorandil group (patients received intracoronary administration of 6 mg nicorandil after guide wire or balloon successfully crossed the target lesion, n=53), the nitroglycerin group (patients received intracoronary administration of 300 μg nitroglycerin after after guide wire or balloon successfully crossed the target lesion, n=52) and the control group(patients received routine treatment, n=53). The primary outcomes were myocardial perfusion, including the levels of corrected TIMI frame count (cTFC), and the incidence of no reflow or slow flow after PPCI. The secondary outcomes included the incidence of major adverse cardiovascular events (MACE) during hospitalization (all-cause death, reperfusion arrhythmia within 2 hours after PPCI, angina within 24 hours after PPCI, new heart failure or worsening cardiac function, and repeat revascularization) and within 3 months of follow-up (all-cause death, nonfatal myocardial infarction, repeat revascularization, post-infarction angina, and re-hospitalization for congestive heart failure). Results: The age of enrolled patients was (62.9±11.3) years old, and 130 cases (82.3%) of them were male. The median time of symptom-onset to balloon was 4.50 (3.20, 6.43) hours. There were significantly difference in cTFC immediately after PPCI((21.68±7.43)frames, (24.74±8.66)frames, and(27.06±10.40)frames), incidence of no reflow or slow flow after PPCI(5.7%(3/53), 13.5%(7/52), and 22.6%(12/53)), ST-segment resolution at 2 hours after procedure(90.6%(48/53), 84.6%(44/52), and 74.5%(38/53)), and reperfusion arrhythmia at 2 hours after procedure(15.1%(8/53), 36.6%(19/52), and 34.0%(18/53)) among the 3 groups(P<0.01 or 0.05). In the multivariate logistic regression models, intracoronary administration of nicorandil could lower the cTFC level (OR=0.17, 95%CI 0.10-0.41, P=0.001), acted as a protecting factor on lowering the incidence of no reflow or slow flow (OR=0.13, 95%CI 0.02-0.96, P=0.045) and reperfusion arrhythmia (OR=0.26, 95%CI 0.09-0.74, P=0.012), as well as facilitating the ST-segment resolution at 2 hours after procedure (OR=4.62, 95%CI 1.14-18.82, P=0.033). However, observed parameters were similar between intracoronary administration of nitroglycerin group compared with control group (all P>0.05). MACE within 3 months of follow-up were similar among the 3 groups(all P>0.05). Conclusion Intracoronary administration of nicorandil prior to balloon dilation can significantly improve the myocardial perfusion and reduce the occurrence of reperfusion arrhythmia during PPCI for STEMI, but does not affect the short-term prognosis in STEMI patients.

Different endophytes isolated from the seeds of Sophora flavescens were tested for their ability to produce matrine production. Response surface methodology (RSM) was applied to optimize the medium components for the endophytic fungus. Results indicated that endophyte Aspergillus terreus had the ability to produce matrine. The single factor tests demonstrated that potato starch was the best carbon source and the combination of peptone and NH₄NO₃ was the optimal nitrogen source for A. terreus. The model of RSM predicted to gain the maximal matrine production at 20.67 µg/L, when the potato starch was 160.68 g/L, peptone was 24.96 g/L and NH₄NO₃ was 2.11 g/L. When cultured in the optimal medium, the matrine yield was an average of 20.63 ± 0.11 µg/L, which was consistent with the model prediction. This study offered an alternative source for the matrine production by endophytic fungus fermentation and may have far-reaching prospect and value.
Aspergillus* ; Carbon ; Endophytes ; Fermentation* ; Fungi* ; Nitrogen ; Peptones ; Solanum tuberosum ; Sophora* ; Starch

Objective To inhibit the proliferation and metastasis of colon cancer cells by increasing the expression level of B-cell translocation gene-2 (BTG2).Methods Western blot assay was used to detect the expression level of BTG2 protein in the normal intestinal epithelial HIEC cells and three colon cancer cell lines SW620, HT-29 and LS174T.The expression of BTG2 protein in normal colonic epithelial tissue, colon adenoma and colon cancer tissue was detected by immunohistochemistry.The plasmid with BTG2 gene full-length sequence was transfected into colon cancer SW620 cells, and the expression of BTG2 protein was detected by Western blot.The cell growth curve was drawn by MTT test.The Ki-67-positive rate was calculated using immunofluorescence staining.The cell migration of colon cancer cells was detected by scratch test and Transwell double chamber culture system, and the pseudopodia growth of tumor cells was detected by Matrigel 3D culture system.Results Western blot results showed that BTG2 relative expression levels were 0.83±0.12,0.18±0.04, 0.20±0.05 and 0.36±0.07 in normal human intestinal epithelial cells HIEC, and human colon cancer cell line SW620, HT-29 and LS174T, respectively.The results of immunohistochemistry showed that the positive expression of BTG2 protein in normal colorectal tissue, colorectal adenoma and colorectal carcinoma tissues were 82.5%(33/40), 77.5%(31/40) and 17.5%(7/40), respectively, with a significant difference between two groups (P<0.05).Immunofluorescence results showed that the positive rate of Ki-67 in the control group, empty vector group and BTG2 transfection group was (76.2± 8.0)%, (81.4±9.7)%and (50.1±7.1)%, respectively, showing a significant difference between two groups ( P<0.05 ) .The scratch test results showed that in the control group, empty vector group and BTG2 transfection group , the distance of SW620 cells between two sides was ( 79.27 ±11.24) μm, ( 80.65 ± 12.17) μm and (124.77±19.63) μm, respectively, with a significant difference between two groups ( P<0.05) .Transwell results showed that in the control group, empty plasmid group and BTG2 transfection group, the SW620 cell migration rate was (78.5±13.1)%, (73.2±12.9)%and (47.4±9.1)%,respectively, showing a significant difference between two groups ( P<0.05) .The number of neurospheres of BTG2 transfection group was decreased SW620, which had poor ductility.Conclusion s BTG2 gene is involved in colon cancer cell proliferation and metastasis , and effectively restores the function of BTG2 protein.Therefore, it may be expected to become a new option in gene therapy for colon cancer.

Objective To inhibit the proliferation and metastasis of colon cancer cells by increasing the expression level of B-cell translocation gene-2 (BTG2).Methods Western blot assay was used to detect the expression level of BTG2 protein in the normal intestinal epithelial HIEC cells and three colon cancer cell lines SW620, HT-29 and LS174T.The expression of BTG2 protein in normal colonic epithelial tissue, colon adenoma and colon cancer tissue was detected by immunohistochemistry.The plasmid with BTG2 gene full-length sequence was transfected into colon cancer SW620 cells, and the expression of BTG2 protein was detected by Western blot.The cell growth curve was drawn by MTT test.The Ki-67-positive rate was calculated using immunofluorescence staining.The cell migration of colon cancer cells was detected by scratch test and Transwell double chamber culture system, and the pseudopodia growth of tumor cells was detected by Matrigel 3D culture system.Results Western blot results showed that BTG2 relative expression levels were 0.83±0.12,0.18±0.04, 0.20±0.05 and 0.36±0.07 in normal human intestinal epithelial cells HIEC, and human colon cancer cell line SW620, HT-29 and LS174T, respectively.The results of immunohistochemistry showed that the positive expression of BTG2 protein in normal colorectal tissue, colorectal adenoma and colorectal carcinoma tissues were 82.5%(33/40), 77.5%(31/40) and 17.5%(7/40), respectively, with a significant difference between two groups (P<0.05).Immunofluorescence results showed that the positive rate of Ki-67 in the control group, empty vector group and BTG2 transfection group was (76.2± 8.0)%, (81.4±9.7)%and (50.1±7.1)%, respectively, showing a significant difference between two groups ( P<0.05 ) .The scratch test results showed that in the control group, empty vector group and BTG2 transfection group , the distance of SW620 cells between two sides was ( 79.27 ±11.24) μm, ( 80.65 ± 12.17) μm and (124.77±19.63) μm, respectively, with a significant difference between two groups ( P<0.05) .Transwell results showed that in the control group, empty plasmid group and BTG2 transfection group, the SW620 cell migration rate was (78.5±13.1)%, (73.2±12.9)%and (47.4±9.1)%,respectively, showing a significant difference between two groups ( P<0.05) .The number of neurospheres of BTG2 transfection group was decreased SW620, which had poor ductility.Conclusion s BTG2 gene is involved in colon cancer cell proliferation and metastasis , and effectively restores the function of BTG2 protein.Therefore, it may be expected to become a new option in gene therapy for colon cancer.