| PurposePeople are inereasedly realized the hazard of smoking passively or active acting on fetus. All these owe to nicotine, one of twenty kinds of poisonous compositions, which can enter fetus body through placenta and make the embryo slowly developed, malformed and aborted. Nicotine is determined to result in the symptom mentioned above. The embryo during pregnancy is susceptible to different of nicotine.Nicotine, as a colorful, easy - volatized, toxic easy to enter the placenta, which will do harm to the fetus, even lead to a under - developed neural tube no matter smoking passively or actively, the response of mouse brain to the environment is quite sensitive.More and more attention had been paid on relation between under - developed neural tube and smoking. Joschko MA said that nicotine may lead to the epithelium death in keeping with the concentration of nicotine, which can draw a conclusion that nicotine, as a of nerve system, will lead to a abnormal morphology. Tara Sankar Roy took the technology of the whole embryo culture to incubate mouse embryo in medium with the different concentration of nicotine. And observe the extensive appearance of cell damage on protocerebrum, deutocerebrum and tritocerebrum such as more vacuole, the increasing number of damage epithelium and larger space. Wang observed a irregular arrange of the neural epithelium and more vacuole in cytoplasm when exposed to nicotine. And so he drew a conclusion that the nicotine will induce a cell damage and loss which subsequently results in the defect of formation and differential process.This study take nestin and S - 100β, as the marker of epithelium before andafter differentiation,to observe the morphology of E13 neural tube and E16,and differentiation of neural epithelium exposed to nicotine during mid phase of pregnancy , and provide the support of theory about the comprised neural tube exposed intrauterinely to nicotine during pregnancy.The enclosed neural tube will subsequently develop to brain, spinal cord and other neural tissue. Once neural tube enclosed abnormally, the subsequent development of fetus including nerve system, skeleton system and muscle tissue may not fall into problem. These congenital disease are called Neural Tube Defect baby. The born of these babies will bring burden to family and society. So the topic on the effect of nicotine on the development of neural tube is not only a medicine problem but also a social one deserved to pay more concentration to.Methods1. Establishment of animal model that intrauterinely exposed to nicotine during pregnancyKunming mice of closed HErd, mate by (?) : (?) = 2: 1, cHEck pessus or sperm 2 times every day and the day that pessus or sperm ( + ) represents start of pregnancy ( marked with E0) , and the pregnant mouse be feed separately. The pregnant mice are divided into 2 groups: test ( NIC) group and control ( CON) group. From the day of E4, NIC group accept 2 subcutaneous injections of nicotine totally 3. 0 mg/kg every day, and the same volumes of saline for CON group until parturition.2. HE staining result of neural tube tissue change of E13 mouse embryo and midline of P16 mouse embryoMouse embryo on E13 or E16 are perfused with 4% formaldehyde, 10um sections are made; wash the section 5min,hematoxylin staining for 3min; wash with tap water for 5min ; 1% hydrochloric acified ethanol deal for 5sec, water wash for 5 min; eosin staining for 1 min, wash for another 5 min; dehydrate, hyalin-ize and seal sections.3. Immunohistochmistry result of neural tube tissue of E13 mouse embryo and midline of E16 mouse embryo.Cells are washed in PBS for 3 times and fixed in 4% formaldehyde, hyalin-ized in 0.4%TritonX -100, blocked by normal serum albumin, incubated with the first antibody for 1h at 37℃ ,the second antibody for 1h in room temperature (in darkness for immunofluorescence) , 1 * PBS wash 5 minute,3 times between beyond steps. Finally seal cell and then observe under microscope.4. Establishment of cell model exposed to nicotine and immunocytochemis-trya. Neural epithelium in vitro cultureEmbryos are removed from the pregnant mouse on embryonic day 18, the midbrain of 5 - 8 embryos of was dissected and washed twice in D - Hank' s buffer, digested in preheated trypsin (0. 25%) , After 30min of incubation, stop digestion by F12(5%FBS) , centrifuge at 500 x g, the cells are resuspend-ed and counted under microscope, after appropriate dilution the cells are plated onto cover slips previously coated with poly - D - lysine and After incubated for 2 hours in 5%CO2,37℃ , add 2ml culture medium F12(5%FBS) including the different concentration(1,10, 100μmol/L) of nicotine into the dish, change medium after 48hours till the 5th incubation.b. cell immunocytochemistry:Cells are washed in PBS for 3 times and fixed in 4% formaldehyde, hyalin-ized in 0.4%TritonX -100, blocked by normal serum albumin, incubated with the first antibody for 1h at 37℃,the second antibody for 1h in room temperature (in darkness for immunofluorescence) , 1 * PBS wash 5 minute,3 times between beyond steps. Finally seal cell and then observe under microscope.5. HE result of neural epithelium in vitro cultureProliferation comparison of the neural epithelium under different concentration of nicotine by MTT colorimetry.Add 0.1 ml MTT medium into 96 well dish and pat repeatly. After 4 hours of incubation at 37℃ , put 0. 1ml of acidified dimethylsulfoxide in dish till the homogenous blue medium appeared. Evaluate value by enzymetic equipment at 570nm.Results1. Establishment of the mouse model exposed to nicotine during pregnancy.1. 1 Six pregnant mice were got separately and feed individually from group NIC and CON. No distinct different of body weight increase was found after nicotine injection; embryo was got out on E13 and E16; For E13 pregnancy mouse, 36 embryos of 3 pregnant pups from nicotine group and 42 of control group were got, 3 and 1 dead embryo separately were observed; For E16 pregnancy mouse, 30 embryos of 3 pregnant pups from nicotine group and 34 embryos from control group were got and 6 and 2 dead embryos were observed.1.2 Result of stereoscope:Near the fourth brain of midbrain in CON group of E13, we can observe a membrane exists, which is the roof floor of neural tube, whereas about 20 embryos per 36 were not observed in NIC group.2. HE result2.1 Roof and floor of neural tube for nicotine and control group of E13 mouse embryoAlthough the covered neural tube was observed in both groups, no neural epithelium covers part of neural tube in nicotine group, as well as the floor plate.2.2 HE result of midline tissue of E16 embryo: the midline zone could come into being when the hemisphere converges. The part of midline zone in nicotine group was not close completely whereas the control was completely occluded.3. Result of immunohistochemistry3.1 Weaker expression of nestin on the roof of E13 mouse embryo in nicotine group was observed, and no neural epithelium covered part of region.3.2 No difference was observed for nestin expression on the midline zone of E16 embryos. But less expression of S - 100βcould be observed.4. Establishment of cell model exposed to nicotine 4.1 Result of neural epithelium in vitro culture... |
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