PURPOSE:To establish methods for cryopreservation of human retinal pigment epithelial cells (RPEs)and cell culture from thawing of frozen cells. METHODS:Primary cultured RPEs or its first or second passages,added with 10 dimetbylsulfoxide,were kept in --20℃ for 1 to 2 hours,and then further froze to -40~C over night before being placed in liquid nitrogen. The frozen cells were thawed in 60℃ within 2 minutes. Trypan blue staining and immunocytochemical staining with anti-human keratin were performed for cell viability and differentiation. The growth curve was also determined by calculating the total number of cells/well/day. RESULTS:The viable rate from frozen RPEs was 90%. No differences were observed for growth activity between cultures from frozen cells and controls. The cells were positive with anti-human keratin staining. The logarithmic growth phase was during I to 4 days and the doubling time yeas 1.55 days. CONCLUSION: Cryopreservation of RPEs in liquid nitrogen can maintain biological activities of cells with normal growth and features after thaw- ing. This will provide cell lines for in vitro experiments and possibly for cell banks for RPE transplantation for some fundus diseases. （Chin J Ocul Fundus Dis,1997,13:157-159）
Abstract In order to supply allografts for reconstruction of finger, repair of tendon or tendon sheath, the hands from fresh cadavers of healthy young persons who died from accident were amputated at the wristlevel. After disinfected, packed and labelled, the hands were stored into a deep freezer at -30℃ as bank storage. Before grafting, the skin and subcutaneoustissue were stripped from the frozen finger. Then it was immersed in an antibiotic fluid for 1 hour. The results showed that the immunological antigenicity of allograft was decreased by the freezing method. Bony union between the impacted host bone and the allogeneic phalangeal bone was seen on X-ray films. Bone absorption and joint degeneration were found at the area where no impaction between bones. The healing between tendons from host and that from the allografts was b. It was concluded that establishing a cadaveric hand bank in hospital was as important as establishing a bone bank for the supply of bone, joint, tendon, tendon sheath and finger composite tissue, for allogeneic grafts.
In order to repair cartilage defect in joint with transplantation of cryopreserved homologous embryonic periosteum, 30 rabbits were used and divided into two groups. A 4 mm x 7 mm whole thickness cartilage defect was made in the patellar groove of femur of each rabbit. The homologous embryonic rabbit skull periosteum (ERSP), preserved in two-step freezing schedule, was transplanted onto the cartilage defect of joints of one group and autogenous periosteal graft was done in the joint defect of the other group. The knees were not immobilized, following operation and 16 weeks later, the newly formed tissue in the defects were assessed by gross observation, histochemical examination and biochemical analysis. The results showed that new hyaline-like cartilage was formed in the cryopreserved ERSP grafted knee, and had no significant difference from that of the knee receiving autogenous periosteal graft, but had significant difference from that of the fresh ERSP grafted knee and the non-grafted knee. Furthermore, the new hyaline-like cartilage had the biochemical characteristics of a fibrous cartilage. The conclusion was that this method might be feasible to repair articular cartilage defects.
Objective To evaluate which is better method zymogen or low temperature frozen in removing vascular endothelial cell so as to lay a foundation for creating a kind of brace which is not to be rejected and the same as own blood vessel. Methods Fresh and not damaged umbilical blood vessel was collected from natural labour women, human umbilical blood vessel was remove carefully from normal foetus, then was put into disinfectant at 37℃ for 24 hours. They were divided into 3 groups:normal group(NG),zymogen group(ZG) and low temperature frozen group(LG). ZG: 0.1% collagenⅡ enzyme was addedin umbilical blood vessel and closed the both sides and the vascular endothelialcell was removed in 37℃ water. LG:Umbilical blood vessel was put into liquidnitrogen for 24 hours after frozened step by step, and then it was put into 37℃ water for 30-60 s and the vascular endothelial cells were washed away by normal saline. NG:Umbilical blood vessel was kept into 4℃ Kerb’s liquid. The bacteria were culturedin each group. The samples were stained by HE,elastic fiber and collagen fiberwere observed by light and scanning electron microscope. The difference of compliance was compared. Human leukocyte antigen ABC(HLA-ABC) and HLA-DR were observed by immunohistochemical method and the expression of antigen of umbilical blood vessel was analysed. Results In LG, umbilical vascular endothelial cells were removed completely; artery showed vertical smooth muscle and vein showed elastic membrane. InZG, umbilical vascular endothelial cells were removed completely after 20 minutes;artery showed vertical smooth muscle cells and vein showed lower endothelial layer. The vascular compliance in LG was higher than that in NG, and the latter was also higher than that in ZG,but showing no significant differences (Pgt;0.05). The compliance of umbilical vein was 2-3 times as much asthat of umbilical artery.The expression of HLA-ABC and HLA-DR in LG andZG were lower than that in NG, showing significant differences (Plt;0.01). Conclusion Low temperature frozen methodand zymogen method(0.1% collagen Ⅱ enzyme for 20 min) can remove vascular endothelial cells of human umbilical blood vessel completely.Low temperature frozenmethod was better than zymogen method.
Abstract： Objective To evaluate the clinical safety and neurological outcomes of right axillary artery cannulation with a side graft compared with a direct approachin aortic arch replacement for patients with acute Stanford type A aortic dissection. Methods Between July 2008 and July 2010， 280 consecutive patients with acute Stanford type A aortic dissection underwent right axillary artery cannulation for cardiopulmonary bypass （CPB） in total arch replacement and stented “elephant trunk” implantation in our hospital.These 280 patients were divided into two groups according to the method of axillary artery cannulation in operation:direct arterial cannulation was used in 215 patients（direct arterial cannulationgroup， DG group， mean age of 43.1±9.5 years）， while cannulation with a side graft was used in 65 patients（ indirect cannulation group， IG group， mean age of 44.7±8.3 years）. Clinical characteristics of both groups were similar except their axillary artery cannulation method. Patient outcomes were compared as to the prevalence of clinical complications， especially neurological deficits and postoperative morbidity. Results The overall hospital mortality was 3.6% （10/280）， 3.3% （7/215） in DG group and 4.6% （3/65） in IG group respectively.Right axillary artery cannulation was successfully performed in all cases without any occurrence of malperfusion. Postoperatively， 25 patients（8.9%）developed temporaryneurological deficits， 19 cases in DG group（8.8%）， and 6 cases in IG group （9.2%）， and all these patients were cured after treatment. The incidence of postoperative complications directly related to axillary artery cannulation was significantly lower in IG group than that in DG group（1 case vs. 19 cases， P=0.045）. There were no statistical differences in arterial perfusion peak flow， peak pressure，antegrade cerebral perfusion time， deep hypothermic circulatory arrest time， and CPB time between the two groups（P ＞ 0.05）. Conclusion Right axillary artery cannulation with a side graftcan significantly reduce the postoperative complications of axillary artery cannulation. It is a safe and effective method for patients undergoing surgery for acute Stanford type A aortic dissection.
目的:探讨TCR(低温等离子射频)序贯治疗在治疗阻塞性睡眠呼吸暂停低通气综合征(OSAHS)临床疗效。方法:我院2003年8月至2007年2月收治153例轻中度OSAHS患者,采用TCR序贯治疗,初次手术后追踪患者情况,必要时分阶段分部位反复消融,并在术后半年,1年进行PSG检查等,对其疗效、并发症进行分析。结果:153例患者半年有效率86.27%。1年有效率73.20 %,无严重并发症发生。结论:TCR序贯治疗疗效确切,组织反应轻,可作为治疗轻中度OSAHS的有效方案。
Objective To observe the configuration and viability of full thickness human fetal retina after short-, mid- and long-term preservation. Methods Twenty-two full thickness human fetal retinae of gestational age of 12-24 weeks were coated by glutin and cut into 88 pieces, and then preserved in Ames' solution, DX solution, -80℃ refrigerator or under cryopreservation condition. The cell viability of retinal neuroepithelial layer was determined by trypan blue staining, retinal configuration was determined by light microscope and electromicroscope. Results The viability of neuroepithelial layer was (94.79plusmn;2.85) % in fresh fetal retina, gt;80% in Ames' solution within 4 hours, and gt;77% in DX solution within 2 days. There was no significant difference between those solution-preservations and the fresh fetal. In -80℃ refrigerator, the viability was (65.83plusmn;5.06)% after 7 days, and then dropped to (57.54plusmn;16.18)% at the end of the first month. Under the cryopreservation condition, the viability was (69.46plusmn;9.31)% at the end of first month. Light and transmission electron microscopy had not deteced any abnormals in the full thickness human fetal retina preserved in Ames' solution within 2 hours, but showed clear retinal layers with bigger intercellular space after preserved in DX solution for 2 days, in -80℃ refrigerator for 7 days and under cryopreservation condition for 1 month. Conclusion Ames' solution and DX solution can preserve good viability and configuration of full thickness human fetal retina in a certain time period.