نام فایل : METHOD~1

فرمت : .DOC

تعداد صفحه/اسلاید : 6

حجم : 128 کیلوبایت

Methods
Procurement and culture of the human fetal pancreas
Eight consecutively obtained human fetal pancreatic glands were collected in a period of 2 months at the Department of Obstetrics and Gynecology, the Affiliated Hospital of Luzhou Medical College, Sichuan, China after the approval of the Ethics Committee of the hospital. The gestational age of these fetuses (3 were male and 5 female) varied from 14 to 32 weeks and all abortions were induced with water bag. The pancreas was usually dissected within 2 hours after abortion. All procedures were carried out under sterile conditions in a laminar flow cabinet according to the modified method.[11,12]
 
Blood samples were collected from umbilical cord vessels of 5 healthy and term-pregnancy delivered newborns, whose mothers had no history of any diseases at the time of birth. The samples were centrifuged to separate serum. The pooled serum or UCS was stored at -20
℃
and kept inactivated and aseptic until use.[13]
 
After removal, the intact gland was maintained in Ca
2+
and Mg
2+
-free D-Hank?/SPAN>s solution at 4
℃
for further processing. For tissue culture, the gland was washed repeatedly in 5 ml D-Hank?/SPAN>s solution with 100 U/ml penicillin and 100 ?/SPAN>g/ml streptomycin in glass dishes. Nonpancreatic tissues and loose connective tissue capsules surrounding the gland were also dissected free. The pancreas was successively minced with scissors to make segments of about 1 mm
3
, which were then transferred to glass vials containing 5 ml D-Hank?/SPAN>s solution with 2.5 mg /ml V-type collagenase (528 U/mg, pH 7.2, Sigma, USA). The fragments were shaken rapidly in a water bath at 37
℃
, and incubated in D-Hank?/SPAN>s solution at 4
℃
for 5-10 minutes when they were almost disintegrated and noticed by naked eyes. The tissue suspension was washed twice in 10 ml D-Hank?/SPAN>s solution and centrifuged (500×g for 6 minutes). The pellet was subsequently transferred to a 5 ml serum-free RPMI-1640 medium (Sigma, USA) and filtered through a 108-?/SPAN>m-pore sized nylon mesh. The resulting pellet was resuspended in a 2 ml RPMI-1640 medium and divided into two culture dishes, each containing 3 ml RPMI-1640 (11.1 mmol/L glucose). Finally, 1 ml 10% FCS (Sigma) or 1 ml 10% (vol/vol) UCS was added to each dish. Cultures were performed in incubators at 37
℃
with 95% O
2
/5% CO
...