Objective To study the biological activity of recombinant adeno-associated virus vector (rAAV) coexpressing human vascular endothel ial growth factor165 (hVEGF165) and human bone morphogenetic protein 7 (hBMP-7) genes in vitro so as to provide a new method for the therapeutics of osteonecrosis. Methods The 3rd passage rabbit bone marrow mesenchymal stem cells (BMSCs) were transfected with rAAV-hVEGF165-internal ribosome entry site (IRES)-hBMP-7(experimental group) and green fluorescent protein (GFP) labeled rAAV-IRES-GFP (control group). The expressions ofhVEGF165 and hBMP-7 were detected by ELISA assay at the 1st, 2nd, 3rd, 7th, 14th days and Western blot assay at the14th day after transfection. The expression consistencies of hVEGF165 and hBMP-7 were observed by immunofluorescence assay at the 14th day after transfection. The biological activity of hVEGF165 was assessed by angiopoiesis experiment of the 3rd passage human umbil ical vein endothel ial cells (HUVEC). The biological activity of hBMP-7 was assessed by mineral ization of BMSCs detected by ALP staining and al izarin red staining. Results With infecting time, the hVEGF165 and hBMP-7 expressions increased gradually in two groups, showing significant difference between two groups (P lt; 0.05). The expressions of hVEGF165 and hBMP-7 were positive in experimental group and negative in control group, respectively. Immunofluorescence assay showed positive expressions of hVEGF165 and hBMP-7 in the exprimental group and negative expression in the control group, the expression of hVEGF165 and hBMP-7 had good consistencies. hVEGF165 secreted from BMSCs enhanced HUVEC migration, prol iferation and tube formation in experimental group. There was significant difference in the number of blood vessel between two groups (P lt; 0.05). The ALP staining showed more bly stained granules in experimental group than in control group. There was significant difference in the number of the mineral ized nodules between two groups (P lt; 0.05). Conclusion The rAAV-hVEGF165-IRES-hBMP-7 has good biological activity in vitro.
Objective To investigate an effect of differenttemperature cryopreservation of the two-step freezing method on the Schwann cell biological activity in the peripheral nerve of the rat. Methods Eighty femaleSD rats were randomly divided into 8 groups of 10 rats each. One was the control group and 7 were the experimental groups. Two 2-cm-long sciatic nerve segments were respectively taken from both legs of each rat. In the control group, the sciatic nerve segments did not undergo the treatment of cryopreservation; however, in the 7 experimental groups, the sciatic nerve segments respectively underwent the different temperature cryopreservation of the twostep freezing method at -20℃, -30℃, -40℃, -50℃, -60℃, -70℃ and -80℃. The sciatic nerve segments were cryopreserved for 2 hours，and then placed into the liquid nigrtrogen at -196℃. After 48 hours of storage，the nerve segments werethawed quickly in the 37℃ water bath box for 1 minute. Then, the sciatic nerve segments each group were harvested. The cells of the sciatic nerve were incubated with Calcein-AM for 15 minutes. The average fluorescence intensity of the cells was measured by the flow cytometry. The nerve fibers were also incubated with Calcein-AM for 15 minutes. The fluorescence intensity of the cells was analyzed by the confocal fluorescence microscope. The Schwann cell biological activity intensity was measured. Results The fluorescence intensity in the -40℃ group was the best and the Schwann cell biological activity in this group was thebest among all the groups（P＜0.01）. The fluorescence intensity in the 8 groups measured by the flow cytometry was as follows：242.522 0±9.568 4 in the control group，168.677 0±10.207 0 in the -20 ℃ group，214.992 0±8.329 1 in the -30 ℃ group，235.526 0±9.280 5 in the -40 ℃ group，222.434 0±8.515 5 in the -50 ℃ group，217.409 0±9.515 7 inthe -60 ℃ group，132.376 0±13.459 7 in the -70 ℃ group， and 108.132 0±16.033 1 in the -80 ℃ group. The fluorescence intensity detected by the confocal fluorescence microscope was as follows：143.700 0±5.567 8 in the control group，119.700 0±5.161 5 in the -20 ℃ group，121.300 0±4.347 4 in the -30 ℃ group，700 0±5.012 2 in the -40 ℃ group，121.000 0±4.546 1 in the -50 ℃ group，118.400 0±4.9261 in the -60 ℃ group，81.200 0±5.116 4in the -70 ℃ group，and 79. 000 0±5.716 4 in the -80 ℃ group. Conclusion The Schwann cell biological activity treated by the two-step freezing methodcan be preserved and the activity is cryopreserved best at -40 ℃.
ObjectiveTo optimize HSP65-MUC1 fusion protein purification in pilot scale through protein purification techniques and identify the methods for biological activity detection. MethodsE. coli expressing HSP65-MUC1 was obtained by fermentation, then homogenized to obtain the supernatant. To acquire high-purity, high-quality HSP65-MUC1, the supernatant was treated with saturated ammonium sulfate, phenyl sepharose FF column and Q FF ion-exchange chromatography column purification. The expression of CD86 on the surface of DC cells treated with HSP65-MUC1 was determined with flow cytometry. ResultsE. coli containing pET28a-HSP65-MUC1 recombinant plasmid can effectively express target protein. A total of 413.7 mg of HSP65-MUC1 was obtained after 10 g of fermented cells was treated with saturated ammonium sulfate, phenyl sepharose FF column and Q FF ion-exchange chromatography column, and the purity was nearly 96%. Compared with negative control (10.13%±0.89%), purified HSP65-MUC1 could significantly improve the expression of CD86 on the surface of DC cells (29.98%±1.02%). ConclusionThe pilot scale production of purified HSP65-MUC1 has been effectively optimized, and the methods of its biological activity detection have been identified, which simultaneously provides the basis for clinical studies.
The surface morphology of titanium metal is an important factor affecting its hydrophilicity and biocompatibility, and exploring the surface treatment strategy of titanium metal is an important way to improve its biocompatibility. In this study, titanium (TA4) was firstly treated by large particle sand blasting and acid etching (SLA) technology, and then the obtained SLA-TA4 was treated by single surface treatments such as alkali-heat, ultraviolet light and plasma bombardment. According to the experimental results, alkali-heat treatment is the best treatment method to improve and maintain surface hydrophilicity of titanium. Then, the nanowire network morphology of titanium surface and its biological property, formed by further surface treatments on the basis of alkali-heat treatment, were investigated. Through the cell adhesion experiment of mouse embryonic osteoblast cells (MC3T3-E1), the ability of titanium material to support cell adhesion and cell spreading was investigated after different surface treatments. The mechanism of biological activity difference of titanium surface formed by different surface treatments was investigated according to the contact angle, pit depth and roughness of the titanium sheet surface. The results showed that the SLA-TA4 titanium sheet after a treatment of alkali heat for 10 h and ultraviolet irradiation for 1 h has the best biological activity and stability. From the perspective of improving surface bioactivity of medical devices, this study has important reference value for relevant researches on surface treatment of titanium implantable medical devices.