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）
Liver transplantation plays a pivotal role in the field of treating end-stage liver diseases. It is currently the only treatment method that can effectively save patients’ lives and has been fully verified as effective in clinical practice. However, the problem of postoperative infection has become one of the key factors impeding the favorable prognosis of patients. Among them, donor-derived infections, due to their uniqueness and complexity, are gradually becoming a research hotspot in both clinical and scientific research fields. Through a systematic and comprehensive analysis of a large number of existing studies, we deeply analyzed the overall current situation of infections after liver transplantation, clarified the specific types of donor-derived infections and their respective characteristics. On this basis, the potential risks of such infections were thoroughly explored, and preventive strategies such as the prophylactic use of antibiotics and vaccination were put forward in a targeted manner, aiming to improve the prognosis of patients after liver transplantation to the greatest extent and enhance the quality of life of patients.
Objective To investigate the protective effects of metallothionein （MT） for immature myocardium and myocardial interstitium.Methods Twenty-four rabbits （aged 14-21 days） were divided into 4 groups with random number table, 6 each group. Distilled water was injected intraperitoneally in control group and 3.6% ZnSO4 （1.5 ml/kg） was injected intraperitoneally in group 1, group 2 and group 3. Control group, group 1, group 2 and group 3 isolated working rabbit heart model were used in 24h, 12h, 24h and 48h after intraperitoneally respectively. The MT content, recovery of hemodynamics, biochemistry and myocardial ultrastructure were tested. Results The MT content, hemodynamics recovery, adenosine triphosphate （ATP） content, superoxide dismutase activity, Ca2＋-ATPase activity, synthesizing ATP activity of mitochondria, hydroxyproline in group 2 and group 3 were higher than those in control group and group 1 （P 〈 0. 01）. The myocardial watery content, creatine kinase and dehydrogenase leakage, malondialdehyde content, endothelin, mitochondrial Ca2＋ content in group 2 and group 3 were lower than those in control group and group 1（P〈0.01）. The myocardial ultrastructure injuries were less in group 2 and group 3 than that in control group and group l. Conclusion This study demonstrates that myocardial MT prolonged expression can be induced by ZnSO4 and the ischemia-reperfusion injury of immature myocardium and myocardial interstitium could be reduced by MT.
OBJECTIVE: To investigate the repairing effect of transplantation of allogeneic fetal bone in combination with a covering cryopreserved periosteal allograft to bone defect. METHODS: Twenty Long-eared white male rabbits were chosen as experimental model of bilateral 12 mm combined bony and periosteal radial defect. Cryopreserved allograft periosteum with allogeneic fetal bone were implanted in the left defect as experimental side and fetal bone was simply transplanted in the right defect as control side. Bone repair process in the two groups were compared by macroscopy, microscopy, roentgenograms and the contents of calcium and phosphate in the defect area at 2, 4, 8 and 12 weeks after transplantation. RESULTS: There was significant statistic difference in the contents of calcium and phosphate between the experimental and control sides at 4, 8 and 12 weeks after transplantation (P lt; 0.05). With time passing by, the contents of calcium and phosphate have the increasing trends. In the experimental group, lamella bone was seen and medullary canal recanalized at 8 weeks postoperatively. The histological section showed the bone lacuna and lamella bone were formed. CONCLUSION: It suggests that allogeneic fetal bone in combination with a covering cryopreserved periosteal allograft can promote bone repair, and allogeneic fetal bone is excellent bone substitute.
Abstract： Objective To assess the effects of simultaneous antegrade/retrograde cardioplegia (SARC) on myocardial perfusion and energy metabolism in ischemic myocardium using magnetic resonance imaging (MRI). Methods After the hearts were harvested from 18 domestic pigs, left anterior descending artery, aorta, anterior ascending cardiac vein and coronary sinus were cannulated to establish the perfusion routes. 6 hearts were used to assess the effects of SARC on myocardial perfusion. Energy metabolism was observed in the other 12 pig hearts. MRI was used to monitor the distribution of contrast agent (gadoliniumdiethylenetriamine penlaacetic acid, Gd-DTPA) in the myocardium after its injection through arterial and retrograde perfusion routes. The efficacies in sustaining myocardial perfusion and energy metabolism were evaluated by using phosphorus-31 magnetic resonance spectroscopy (31P MRS) during antegrade cardioplegia (AC) and SARC respectively. Results It was found that injection of Gd-DTPA into the aorta during AC did not result in signal increase in the ischemic myocardium on MRI. During SARC, however, Gd-DTPA was found in the ischemic region as well as in the other regions, no matter if it was given into the aorta or into the coronary sinus. Moreover,31P spectra showed that occlusion of LAD during AC resulted in severe decrease of the levels of phosphocreatine (PCr) and adenosine triphosphate (ATP), while the level of inorganic phosphate (Pi) increased in LAD-support myocardium. The abnormal metabolic changes were completely abolished by use of SARC. Conclusion It is concluded that SARC can deliver cardioplegic solution to the myocardium distal to a coronary occlusion and can sustain normal energy metabolism in the jeopardized myocardium.
Objective To investigate efficacy and safety of laparoscopic lymphadenectomy combined with pelvic autonomic nerve preservation in patients with rectal cancer. Methods Seventy-three patients underwent D3 radical resection of rectal cancer with pelvic autonomic nerve preservation from March 2015 to October 2016 in the People’s Hospital of Pengzhou City were collected, then were divided into a laparoscopic surgery group (38 cases) and an open surgery group (35 cases) according to the choice of operation. The intraoperative and postoperative indexes were compared between these two groups, and the urination and sexual functions at preoperation and on month 1 after operation for male patient with rectal cancer were evaluated. Results ① The age, gender, tumor diameter, TNM stage, and tumor location had no significant differences between these two groups (P>0.05). ② Compared with the open surgery group, the intraoperative blood loss was less (P<0.05), but the operation time was longer (P<0.05) in the laparoscopic surgery group. The number of lymph node dissection had no significant difference between these two groups (P>0.05). ③ The anal exhaust time and hospitalization stay in the laparoscopic surgery group were significantly shorter than those in the open surgery group (P<0.05), and the incidences of postoperative infection, intestinal adhesion, and intestinal obstruction were significantly lower in the laparoscopic surgery group as compared with the open surgery group (P<0.05). ④ The IPSS score of urination function and the IIEF-5 score of sexual function on month 1 after operation had no significant differences between these two groups (P>0.05), but compared with the preoperative points, the IPSS score was significantly increased, the IIEF-5 score was significantly decreased in the same group (P<0.05). Conclusion The preliminary results of limited cases in this study show that laparoscopic lymphadenectomy combined with pelvic autonomic nerve preservation in patients with rectal cancer is safe and effective, it has some advantages of mild invasion, lower complication rate, and fast covery.
ObjectiveTo investigate the value of ligament remnant preservation during anterior cruciate ligament (ACL) reconstruction by observing the integrity, the tension, the synovial membrane covering, and the color of the reconstructed ligament under arthroscopy. MethodsBetween January 2011 and December 2013, 122 patients who underwent ACL reconstruction and arthroscopic internal fixation removal at 1 year after reconstruction were included in this study. Of these cases, 61 cases underwent ACL reconstruction using the remnant-preserved technique (preservation group);the other 61 cases underwent ACL reconstruction using non remnant-preserved technique (non preservation group). There was no significant difference in gender, age, injury side, body mass index, type of injury, the time from injury to reconstruction, and the result of KT-2000 examination between 2 groups (P<0.05). The reconstructed ACL were observed under arthroscopy when internal fixation was removed, and the effectiveness was evaluated according to the criteria of AO Yingfang. ResultsIn preservation group, the results were excellent in 34 cases, good in 22 cases, fair in 4 cases, and poor in 1 case;and in non preservation group, the results were excellent in 29 cases, good in 20 cases, fair in 10 cases, and poor in 2 cases;and there was no significant difference between 2 groups (Z=-1.320, P=0.187). ConclusionIn ACL reconstruction, the remnant-preserved technique is not obviously better than non remnant-preserved technique in the integrity, tension, membrane covering, and color.
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 investigate the effects of different temperatures on the system of in vitro physiological environment fostering limbs. Methods Twenty-four limbs were harvested from 6 adult Bama mini pigs and were randomly divided into 4 groups (n=6) according to different temperatures: limbs were placed in in vitro physiological environment foster-ing limbs at 26℃ (group A), 4℃ (group B), 10℃ (group C), and 18℃(group D). After 12 hours of perfusion, the morphology observation was done for the structure and ultrastructure changes of the skeletal muscle by light microscope and transmission electron microscope. The mRNA levels of tumor necrosis factor α (TNF-α) and interleukin 1β (IL-1β) were detected by real-time fluorescent quantitative PCR (RT-qPCR). Results Histological results showed that the skeletal muscle exhibited mild edema, integrity of the sarcolemma, and occasional perivascular inflammatory cell infiltration in groups B, C, and D, meanwhile, the cells of group C had normal morphology; however, muscle fibers degenerated, muscle cells were seriously damaged, a great number of inflammatory cells infiltrated in the fractured muscle fibers in group A. Transmission electron microscope results showed as follows: the muscle fibers arranged in disorder, and many focal solubility necrosis occurred in group A; the muscle fibers arranged in order relatively and sarcolemma was still intact, with mild swelling and flocculent degenerative mitochondria in group B; a large number of muscle fibers arranged in order and regularity with clear sarcomere in group C; and the muscle fibers arranged in disorder and irregularity and partly dissolved in group D. RT-qPCR results showed that the expressions of inflammatory factor TNF-α and IL-1β mRNA in group A were significantly higher than those in groups B, C, and D (P lt; 0.05); the expressions were significantly lower in groups B and C than in group D, and in group C than in group B (P lt; 0.05). Conclusion In the system of in vitro physiological environment fostering limbs, temperature plays an important role in the preservation of amputated limbs. It is suggested that 10℃ can significantly attenuate the reperfusion-induced skeletal muscle cell injuries in this system.
ObjectiveTo analyze clinical experience and outcomes of bileaflet preservation in mitral valve replace-ment (MVR) for patients with severe mitral regurgitation (MR). MethodsWe retrospectively analyzed clinical data of 17 patients with severe MR who underwent MVR with bileaflet preservation in the Department of Cardiovascular surgery of Guangdong General Hospital from June 2011 to January 2013. There were 14 males and 3 females with mean age of 63.41±11.82 years (range, 38 to 82 years). There were 13 patients with atrial fibrillation. Preoperatively, 5 patients were in New York Heart Association (NYHA) functional class Ⅲ, and 12 patients were in NYHA class Ⅳ. There were 7 patients with ischemic MR, 9 patients with degenerative MR, and 1 patient with rheumatic MR. ResultsMVR with bileaflet preservation was performed for all the patients. Concomitant coronary artery bypass grafting was performed for 4 patients. Eleven patients received bioprosthetic MVR, and 6 patients received mechanical MVR. There was no in-hospital death, postoperative low cardiac output syndrome or left ventricular rupture. All the 17 patients were followed up for a mean duration of 16.44±5.02 months (range, 2 to 25 months). During follow-up, 1 patient died of severe paravalvular leak 2 months after surgery. All the other patients had good mitral valve function. None of the patients had anticoagulation or prosthetic valve related complication. Patient's heart function was significantly improved. Eleven patients were in NYHA functional class Ⅰ, 4 patients were in NYHA class Ⅱ, and 1 patient was in NYHA class Ⅲ. Cardiothoracic ratio, left atrial dimension, left ventricular end-diastolic dimension and left ventricular end-systole dimension postoperatively and during follow-up were significantly smaller than preoperative values. Postoperative left ventricular ejection fraction (LVEF) was significantly lower than preoperative LVEF(50.94%±8.78% vs. 55.31%±10.44%, P=0.04), but LVEF during follow-up was not statistically different from preoperative LVEF(55.31%±10.44% vs. 56.13%±9.67%, P=0.73), and LVEF during follow-up was significantly higher than postoperative LVEF(56.13%±9.67% vs. 50.94%±8.78%, P=0.02). There was no statistical difference between postoperative mitral pressure half-time (PHT)and PHT during follow-up (95.06±19.00 ms vs. 94.56±19.19 ms, P=0.91). ConclusionMVR with bileaflet preservation is a safe and effective surgical technique for patients with severe MR, and can significantly improve postoperative left ventricular remodeling and function.