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.
Objective To observe the enzymic histochemical and ultrastructral changes of cryopreserved human retina. Methods To compare the activity of lactate dehydrogenase (LDH)， succinate dehydrogenase (SDH) and ATPase in cryopreserved retina with those in fresh retina and to observe the histological and ultrastructural changes of cryopreserved retina. Results There was no statistical difference between the activity of LDH，SDH and ATPase in fresh and in cryopreserved retina. Histologically, in the cryopreserved retina, fluid in neural fiber and outer plexiform layers, as well as in cone and rod layer, was sligthly more than normal. The ultrastructure is normal except that the mitochondria was swollen in different degree. Conclusion Cryopreservation may be an effective method for keeping the retinal cells alive for a long period and might free the transplantation from dependance on aviability of fresh dornor tissue. (Chin J Ocul Fundus Dis，2000，16：139-212)
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.
To valuate cerebral protection by retrograde cerebral perfusion (RCP) via superior vena cava,the study results for the last ten years have been reviewed.RCP is regarded as an assistant method in deep hypothermic circulatory arrest(DHCA) in that it provides partial brain blood flow,maintains a low brain temperature,optimizes cerebral metabolic function during DHCA by supplying oxygen and some nutrient and removal of catabolic products;it also reduces the incidence of cerebral embolization by flushing out air...
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）
摘要：目的：探讨低温双极射频消融技术治疗多平面阻塞性睡眠呼吸暂停低通气综合征（obstructive sleep apnea-hypopnea syndrome，OSAHS）的价值。方法：对67例多平面OSAHS患者采用低温双极射频消融治疗，根据术前、术后症状改善情况及多导睡眠监测（polysomnography，PSG）结果的比较判定疗效。结果：67例患者中治愈21例，显效22例，有效15例，无效9例，总有效率86.57%。术前与术后1年AHI和SaO2结果经t检验，差异有显著性(P＜0.01)。所有病例均无并发症发生。结论：低温双极射频消融术治疗阻塞性睡眠呼吸暂停低通气综合征（OSAHS）疗效肯定，特别是同期治疗多平面阻塞安全可靠，具有独特的优势。
Objective To establ ish an animal model of osteonecrosis of the femoral head (ONFH) l ike human. Methods Ten healthy adult three-leg Beagle male dogs weighing (16.0 ± 1.6) kg were conducted as the animal model of ONFH according to the schedule of cryosurgery designed in advance in which l iquid nitrogen, pressurized to 0.5 MPa, was poured into the femoral head for 16.5 minutes. After rewarmed to 0℃ for 10 minutes, the l iquid nitrogen was repoured into the femoral head for another 16.5 minutes. At the end of the follow-up, the results were reviewed by pathologic check. One dog was conducted as control group. Results The first boundary temperature of (—27.9 ± 4.3)℃ was higher than the second boundary temperature (— 31.3 ± 4.7)℃ by —3.4℃ , and there was significant difference (P lt; 0.01). The diameter of the femoral head of (17.7 ± 1.1) mm was l inearly (^ y= — 2.6 － 2.409 x) correlated to boundary temperature by Pearson analysis, and the R rate was —0.977 (Plt; 0.05). Four dogs in experimental group progressed to collapse of the femoral head l ike human in the 6th month after operation. The rate of the femoral head collapse rose to 44.4%. In the control group, osteonecrosis was never found. Conclusion Cryosurgcry for osteonecrosis of the femoral head in the three-leg canine model may become a method to establ ish an animal model of ONFH l ike human.
OBJECTIVE: To investigate a cryophylactic agent (CPA) to protect tissue engineered tendon (TET) in deep low temperature. METHODS: Sixty-four BALB/C inbred nude mice were chosen, which included 4 as blank control group, left sides of 60 as experimental group and their right sides as control group. Transformed human embryonic tendon cells of the 54th passage and artificial materials of carbon fiber (CF) and polyglycolic acid (PGA) were co-cultured in vitro to construct TET. TET was frozen in liquid nitrogen with four kinds of CPA (groups A, B, C, and D) for 2 months. They were thawed quickly and transplanted into hind limbs of nude mice to repair the defects of Achilles tendon, which was 5 mm in length and 65.7% of total Achilles tendon. In control group, no cryopreservation treatment was taken. The morphological, histological, ultrastructure, and immunohistochemistry examinations were made and short tandem repeat loci were detected 2, 4, 6, 8, and 12 weeks later. RESULTS: In the experimental group, the morphological properties of tendon cells resumed gradually and the capability of synthesizing collagen enhanced by degrees. Tendon cells survived and could secret type I collagen and there was less difference between experimental and control groups 12 weeks after transplantation. In group A, vacuole in mitochondrion of tendon cell decreased, tendon cell arranged in order and abundant collagen fibers were found and linked. CONCLUSION: The cryopreservation agent in group A can protect TET in deep low temperature.
Objective To evaluate the feasibility of preservation of arteriesby vitrification and the effectiveness of vitrified arteries as allografts. Methods Sixty rabbits were used in the research. Forty-eight femoral arteries wereharvested from 24 rabbits as the transplanted materials，and 24 femoral arteries were preserved by vitrification, 24 by freezing for 14 days，respectively. Theother 36 rabbits were used as the transplanted subjects，and were divided into three groups， 12 rabbits including 24 femoral arteries per group: Group A(fresharterial autografts), Group B(vitrified arterial allografts) and Group C(frozen arterial allografts). The morphologic changes of arterial grafts were observed macroscopically and histologically. The patent rate of arterial grafts were measured by angiography, and the rabbits were sacrificed on the 14th day, the 30th day, the 60th day and the 120th day after transplantation respectively. Arterial grafts were harvested to observe the morphological changes，and the immunological rejection was evaluated by measuring the ratio of tunica intima and tunica media. The results were compared between these groups. Results Before transplantation，theintegrated rate of Group B was 91.67%，which was significantly better than that of Group C（54.17%, Plt;0.01）. After transplantation, the accumulative patent rate of Group B was 87.50%，which was significantly better than that of Group C（66.67%, Plt;0.05）. There was statistically significant difference in the ratioof tunica intima and tunica media between Group B and Groups A, C（Plt;0.05）.Conclusion The above results show that vitrification does less damage to cells and tissues because of ice-free in the process of cryopreservation. So vitrification can be used to preserve arteries， and the arterial allografts preserved by vitrification are better than those preserved by freezing.