[Isolation, culture and cryopreservation of cells derived from fetal appendages].

OBJECTIVE: To investigate the optimal method for isolation, culture and cryopreservation of cells from fetal appendages, for the purpose of providing viable cells for tissue engineering, cell therapy and gene therapy. METHODS: Trypsin dispersion method was used to isolate cells from human umbilical cord and placenta. The tissues from umbilical cord and placenta were cryopreserved with dimethylsulfoxide (DMSO) in different concentrations. Then the percentage of living cells in thawed tissues, and their micro-structure were observed and compared with fresh tissues under transmission electron microscope. The expression of cell immune phenotype before and after cryopreservation were detected with immuno-histochemistry method. RESULTS: The percentage of living cells in human fresh umbilical cord was 67.0%, while that in cryopreserved umbilical cord was 23.4%, 55.5%, 48.8%, 31.8%, respectively in 5%, 10%, 15%, 20% of DMSO. The percentage of living cells in cryopreserved tissues was similar to that of fresh tissues when the volume percentage of DMSO was 10% (P > 0.05), and it was significantly different with that when volume percentage of DMSO was 5% and 20% (P < 0.01). The result by transmission electron microscope was coincident with the results shown above. The results were similar between placenta and umbilical cord. There was no obvious changes in immune phenotype of the tissue and cells after cryopreservation. CONCLUSION: Cryopreservation with this method can isolate a large amount of cells from fetal appendages, with no changes in immune phenotype after cryopreservation, and the effect was best when the volume percentage of DMSO was 10%.

[Changes in the calcitonin content after inhalation injury in dogs].

OBJECTIVE: To investigate the changes in the serum content of immunoreactive calcitonin (iCT) after burns or inhalation injury, and to explore its diagnostic significance. METHODS: Twenty-four dogs were randomized into 4 groups, i. e. A (n = 6, with moderate degree inhalation injury) , B ( n = 6, with severe inhalation injury), C (n = 6, with most severe inhalation injury) and D (n = 6, with severe burns) groups. The serum content of iCT and blood gas analysis before and after injury were determined at different time points. The degree of inhalation injury was determined with fibrobronchoscopic examination at 6 post-inhalation injury hour (PIH). RESULTS: (1) Fiber bronchoscopic examination showed that the degree of inhalation injury in each group was coincident with the anticipation. (2) The serum content of iCT in each group at 1 PIH was obviously higher than that before injury, and it was evidently higher in A, B and C groups than that in D group at 4 PIH. The peak value of iCT in group A at 24 PIH was (453+/-224) ng/L, and it increased gradually in B and C groups at 48 PIH. The serum content of iCT increased continually from 2 PIH on, and it reached (125+/-41) ng/L at 48 PIH. (3) Compared with PaO2 value before injury (109+/-8) mmHg, there was no obvious difference of the PaO, in A and D groups. PaO2 value in B and C group began to descend continually at 8 PIH (65+/-6) mmHg, and that in C group began to descend at 4 PIH (71+/-9) mmHg. PaCO2 value in C group began to increase at 24 PIH(52+/-11) mmHg when compared with that before injury(38+/-5 ) mmHg. CONCLUSION: The changes in the serum level of iCT within 8 PIH occurred much earlier than PaO2 and PaCO2, thus it has the same diagnostic significance as fibers bronchoscopic examination.

An experimental model of an electrical injury to the peripheral nerve.

OBJECTIVE: Injury to the peripheral nerves is a common complication found in patients suffering from electrical burns. At present, there are many kinds of experimental models for electrical injury, but no report describes an animal-based experimental model for a relatively simple electrical injury to the peripheral nerves. We have designed and constructed a specific device to generate increasingly severe electrical shocks of a known voltage for the experiment. This device can simulate injuries of different degrees (minor, medium and severe) caused by shock to the right sciatic nerve of rats. METHOD: Thirty Sprague-Dawley rats were randomly divided into Group I (3600 V, n=10), Group II (1000 V, n=10) and Group III (500 V, n=10). The voltage required for the electrical shock was generated by the above-mentioned device and was adjusted to deliver 3600, 1000 and 500 V, respectively. The specific voltage, as mentioned above, was delivered three times to the right sciatic nerve of the rats. The shock duration was set to last for 10 ms. The time interval between the shocks was 3 min. Three rats were randomly selected from each group to observe changes in the morphology, electric physiology of the nerve and their histology the first, second and fourth week after injury. RESULTS: All rats survived the injuries. Leg function was partially impaired and swellings occurred on the injured extremity. However, by the second week after the injury the rats had recovered. Digit ulcers were observed by the fourth week after injury in Groups I and II. Neural electric physiology showed that the recovery rate of the neural conduction velocity (RNCV) disappeared in part or in whole immediately after the injury in experimental rats. RNCV recovered up to 65% in Group III and to 7% in Group II by the fourth week after injury, however, RNCV did not recover in Group I at all. Histology showed that blood vessel embolism occurred within the injured nerve. A large number of nerve fibres experienced Waller degeneration while the myelin sheath was vacuolated. The neural plate disintegrated largely by the first week after injury and the myelin sheath disintegrated into a loose structure by the second week after injury in Group I. Group II displayed a similar situation as Group I, wherein some nerve fibres experienced Waller degeneration and disintegration. Regenerative myelin appeared in some rats at about the fourth week after injury. The following changes were seen in Group III: The degree of neural injuries was different. The point of entry of the electric currents showed obvious Waller degeneration and disintegration of the myelin sheath, while some nerves showed a regenerated myelin sheath by the second week after injury. The morphology (such as quantity and diameter) of the injured myelin was basically normal by the fourth week after injury. CONCLUSION: This device can produce controlled injuries to the sciatic nerve giving different degrees of severity (minor, medium and severe), by means of varying the electrical shock voltage and shock duration on the rats. It is a useful model for experimental studies of injuries to peripheral nerves.