| With the development of modern society and the improvement of people’s living standards, drinking has become an essential part of social interaction. Drinking alcohol during pregnancy can cause fetal alcohol syndrome (FAS) and fetal alcohol spectrum disorders (FASD), and developmental abnormality in nervous system is one of the major characteristics. Lead is a potent nerve toxicant, and the developing central nervous system is the major target organ of lead. Prenatal lead exposure can cause adverse effects on many aspects such as brain development, function and behavior.Neural stem cells are immature cells that have the potential of self-renewal and differentiation, and play a key role in the development and maturation of the central nervous system. The self-renewal ability refers to the neural stem cells in an undifferentiated state, and maintaining a constant proliferation of features. Pluripotent refers to the capabilities of neural stem cells under appropriate conditions can be induced to differentiate into virtually all cell types of the nervous system. The lead is ubiquitous in the environment, and drinking is widespread concern social issues. Both alcohol and lead can interfere with neural stem cell self-renewal and differentiation, affecting the normal development and function of the nervous system. This study using neural stem cells isolated from fetal rat neocortex investigated the combined effects of alcohol and lead on the self-renewal and differentiation of neural stem cells and the possible mechanisms to provide a scientific basis for studying its mechanism.Part one Combined Effects of Alcohol and Lead on Self-renewal of Neural Stem Cells from Fetal Rat Cerebral Neocortex1. Combined Effects of Alcohol and Lead on Self-renewal of Neural Stem Cells from Fetal Rat Cerebral NeocortexTrypan blue exclusion assay was used to detect combined effects of alcohol and lead on the viability of neural stem cells. Compared with the control, there was no significant difference (P>0.05) in alcohol or lead groups, there was significant difference (P<0.05, P<0.01)) in all alcohol+lead groups. Compared with alcohol groups at the same dose level, there was anobvious downward trend in alcohol+lead groups, and there was a significant differencc in 25mM+0.1μM,25mM+1μM, 50mM+0.1μM and 50mM+1μM groups (P<0.05, P<0.01). Compared with lead groups at the same dose level, these was no significant change in lead (0.01μM)+alcohol groups (P>0.05); there was a downward trend in lead (0.1μM)+alcohol and lead (1μM)+alcohol groups, and there was a significant difference in 25mM+0.1μM,50mM+0.1μM, and 50mM+1μM groups (P<0.05, P<0.01). There was a synergistic interaction on NSC viability between lead and alcohol.2. Combined effects of alcohol and lead on cell cycle of NSCs isolated from fetal rat neocortexFlow cytometry was used to detect combined effects of alcohol and lead on cell cycle of neural stem cells. The results showed that compared with the control group, the proportion of neural stem cells in G0/G1 phase was significantly increased in μM leadgroup and all lead+alcohol groups (P<0.05, P<0.01). Compared with alcohol groups at the same dose level, there was a significant difference in the proportion of Go/G1 phase cells in all lead+alcohol groups (P<0.05, P<0.01). Compared with lead groups at the same dose level, there was an upward trend in all lead+alcohol groups (P<0.05, P<0.01). There was a synergistic interaction on NSC proportion of Go/Gi phase cells between lead and alcohol.Compared with the control, NSC proportion of S phase in the alcohol group,0.1 and 1 μM lead group and the combined groupssignificantly decreased (P<0.05, P<0.01). Compared with alcohol groups at the same dose level, there was a significant decrease (P<0.01) of NSC proportion in S phase alcohol in alcohol (25mM)+lead groups and 50mM+1μM groups. Compared with lead groups at the same dose level, there was a significant decrease (P<0.05,.P<0.01) of NSC proportion in S phase in alcohol+lead(0.01μM), alcohol+lead(0.1μM) and 25mM+1μM groups. There was a synergistic interaction on NSC proportion of S phase cells between lead and alcohol.Compared with the control, NSC proportion of G2/M phase in the 50mM alcohol group andalcohol (50mM)+lead groups was significantly decreased (P<0.05, P<0.01). Compared with alcohol groups at the same dose level, there was a significant decrease (P<0.05) of NSC proportion in G2/M phase alcohol in alcohol (25mM)+lead (1μM) groups and 50mM+1μM groups; there was no significant change (P>0.05) of NSC proportion in G2/M phase alcohol in alcohol (50mM)+lead groups. Compared with lead groups at the same dose level, there was a significant difference (P<0.05, P<0.01) of NSC proportion in G2/M phase in alcohol+lead(0.01μM), alcohol+lead(0.1μM) and 50mM+1μM groups. There was an antagonistic interaction on NSC proportion of G2/M phase cells between lead and alcohol.3. Combined effects of alcohol and lead on cell cycle related genes of NSCs isolated from fetal rat neocortexReal time fluorescence quantitative RT-PCR was used to detect combined effects of alcohol and lead oncell cycle related genes of neural stem cells.The results showed that compared with the control group, the expression of p16 mRNAwas significantly increased in 0.01 μM leadgroups,50mM groups and 25mM+0.01μM groups (P<0.05, P<0.01). Compared with alcohol groups at the same dose level, the expression of p16 mRNAwas significantly increased in 25mM+0.01μM groups (P<0.01). Compared with lead groups at the same dose level, the expression of p16 mRNAwas significantly increased in 0.01 μM leadgroups (P<0.05,P<0.01). There was a certain synergistic interactionon the expression of p16 mRNA between low-dose lead and low-dose alcohol, and antagonism between low-dose lead and high-dose alcohol.Compared with the control group, there was a significant differenceof the expression of p21 mRNAin 50mM+0.01μM groups.Compared with alcohol groups at the same dose level, there was a significant decreaseof the expression of p21 mRNAin 25mM+0.1μM groups and 25mM+1μMgroups (P<0.05),and the expression of p21 mRNAwas significantly increased in 50mM+0.01μM groups (P<0.05). Compared with lead groups at the same dose level, the expression of p21 mRNAwas significantly increased in 0.01μM groups (P<0.01). theexpression of p16 mRNAwas significantly increased in 0.01 μM lead+ alcohol groups (P<0.05), significant decrease in25mM+0.1μM groups and 25mM+1μM groups(P<0.05).Antagonism was the main effects between low-dose lead and low-dose alcohol, and a certain synergistic interaction between high-dose alcohol and low-dose lead.Compared with the control group, there was anobvious upward trend of the expression of p53 mRNA inalcohol groups and lead groups,there was a significant differencein 0.1 μM groups and 1μM groups alone(P<0.05).Compared with alcohol groups at the same dose level, there was a significant increaseof the expression of p53 mRNAin 25mM+0.01μM groups and 50mM+1μMgroups(P<0.05,P<0.01) Compared with lead groups at the same dose level, the expression of p53 mRNAwas significantly increased in 25mM+0.01μM groups (P<0.05). There was an interaction between lead and alcohol as factorial analysis showedthough the R2 is 0.479. The interaction can be considered as significant difference, and further tests are needed to verify the biological significance.4. Alcohol and joint lead exposure on fetal brain neocortex neural stem cell markersReal time fluorescence quantitative RT-PCR was used to detect combined effects of alcohol and lead onNSC markers like Nestin and Sox2.The results showed that compared with the control group, there was a significant difference of the expression of Nestin mRNAin 50mM groups,25mM+0.01μM groups and 50mM alcohol+lead groups (P<0.05, P<0.01). Compared with alcohol groups at the same dose level, there was no significant difference in combined groups (P>0.05). Compared with lead groups at the same dose level, the expression was significantly decreased in 25mM+ 1μMgroupsand 25mMalcohol combined groups(.P<0.05, P<0.01). There was a certain synergistic interactionon the expression of Nestin mRNA between lead and alcohol.For Sox2 mRNA expression, compared with the control, there was a significant decrease in alcohol groups and 25mM+1μM group (P<0.05, P<0.01). Compared with alcohol group at the same dose level, there was no significant change in alcohol(25mM)+lead groups (P>0.05); there was a significant difference in 50mM+1μM group (P<0.05). Compared with lead group at the same dose level, there was no significant change in alcohol+lead(0.01μM) groups and alcohol+lead(0.1μM) (.P>0.05); there was a significant decrease in 1μM+25mM group (P<0.05). There was no interaction of alcohol and lead on NSC Sox2 mRNA expression.5. Alcohol and Joint Lead Exposure on related gene expression in fetal rat brain neocortex of Wnt pathway in neural stem cellsReal time fluorescence quantitative RT-PCR was used to detect combined effects of alcohol and lead onrelated geneSof Wnt pathway in neural stem cells like C-myc and CyclinDl.The results showed that compared with the control group, there was a significant increase of the expression of C-myc mRNAin lead alone groups and 25mM+0.01μM groups (P<0.05, P<0.01). Compared with alcohol groups at the same dose level, there was a significant increase in lead(0.01μM,1μM)+alcohol groups combined groups (P<0.05, P<0.01). Compared with lead groups at the same dose level, the expression was significantly decreased in 25mM+ 0.01μM groups and alcohol+1μMgroups (P<0.05, P<0.01). Synergistic interaction was the main effects between lead and alcohol, and a certain antagonism between low-dose alcohol and low-dose lead.Compared with the control group, there was a significant increase of the expression of CyclinDl mRNAin 50 mM groups, 1μM lead alone groups and alcohol+1μM groups (P<0.05). Compared with alcohol groups at the same dose level, there was a significant increase in 50mM+1μM groups (P<0.05, P<0.01). Compared with lead groups at the same dose level, there was significant difference in 25mM+ 1μMgroups (P<0.05, P<0.01).There was no interaction between alcohol and lead.Part two Combined Effects of Alcohol and Lead on apoptosis of Neural Stem Cells from Fetal Rat Cerebral Neocortex1. Combined effects of alcohol and lead on cell apoptosis of NSCsisolated from fetal rat neocortexAnnexinv/PI staining was used to detect apoptotic rates of neural stem cells by flow cytometry. Compared with the control group, there was no significant difference (P>0.05) in alcohol or lead groups, there was anobvious downward trend in alcohol+ lead groups,andthere was a significant difference (P<0.05) in 50mM+0.1 μM and 50mM+1μM groups. Compared with alcohol groups at the same dose level, there was anobvious downward trend in alcohol+lead groups, and there was a significant difference (P<0.05) in 25mM+1μM and 50mM+1μM groups. Compared with lead groups at the same dose level, these was no significant change in lead (0.01μM)+alcohol groups (P>0.05); And there was a significant difference in 50mM+0.1μM and 50mM+1μM groups (P<0.05, P<0.01).There was no interaction effects on NSC proportion of normal cells between lead and alcohol.Compared with the control group, there was no significant difference (P>0.05) in alcohol or lead groups on early apoptosis, there was anobvious upward trend in alcohol+lead groups, and there was a significant difference (P<0.05) in 50mM+0.1μM and 50mM+1μM groups. Compared with alcohol groups at the same dose level, there was no significant difference (P>0.05)in alcohol+lead groups. There was no interaction effects on NSC proportion of early apoptotic cells between lead and alcohol.Compared with the control group, there was anobvious upward trend in 0.01 μM alcohol or 0.1μM lead groups on late apoptosis, there was a significant difference (P<0.05)in 0.01 μM alcohol or 0.1 μM lead groups, there was anobvious downward trend in alcohol+lead groups, and there was a significant difference (P<0.05) in alcohol(50mM)+lead groups. Compared with alcohol groups at the same dose level, there was no significant difference (P>0.05)in alcohol+lead groups. Compared with lead groups at the same dose level, there was no significant difference (P>0.05) in alcohol+lead groups. There was an interaction effect on NSC proportion of late apoptotic cells between lead and alcohol.There was an antagonistic effectbetween alcohol (50mM)+lead groups, and there was a synergistic interaction between alcohol (25mM)+lead groups.2. Combined effects of alcohol and lead on NSC apoptosis related genesNeurospheres were collected after 48 h dosing, and real-time RT-PCR was used to detect mRNA expression of anti-apoptotic genes Bcl-2andSurvivinand pro-apoptotic gene Baxand apoptoticeffect gene Caspase3.For caspase 3 mRNA expression, compared with the control, there was no significant difference (P>0.05) in all groups; compared with alcohol groups at the same dose level, there was no significant difference (P>0.05) in alcohol+lead groups; compared with lead groups at the same dose level, there was also no significant difference (P>0.05) in alcohol+lead groups. There was no interaction between alcohol and lead on NSC Caspase 3 mRNA expression.For Box mRNA expression, compared with the control, there was no significant difference (P>0.05) in alcohol groups, lead groups and alcohol+lead groups; compared with alcohol groups at the same dose level, there was no significant difference in alcohol (25mM)+lead groups (P>0.05); there was a significant increase (P<0.05) in 50mM+1μM group. Compared with lead groups at the same dose level, there was no significant difference in alcohol+lead (0.01 μM) groups and alcohol+lead (0.1 μM) groups (P>0.05); there was a significant decrease (P<0.05) in 50mM+1μM group. There was no interaction between alcohol and lead on NSC Box mRNA expression.For Bcl-2 mRNA expression, compared with the control, there was a significant difference (P<0.05) in 50mM alcohol group and alcohol(50mM)+lead groups. Compared with alcohol groups at the same dose level, there was a downward trend in alcohol(25mM)+lead groups, and there was a significant difference in alcohol (25mM)+lead(1μM) group (P<0.05). Compared with lead groups at the same dose level, there was no significant difference in alcohol+lead (0.01μM) groups and alcohol+lead (0.1 μM) groups (P>0.05); there was a significant decrease (P<0.01) in alcohol+lead(1μM) group. There was no interaction between alcohol and lead on NSC Bcl-2 mRNA expression.For Survivin mRNA expression, compared with the control, there was a significant difference (P<0.05, P<0.01) in alcohol groups, lead groups and alcohol+lead groups. Compared with alcohol groups at the same dose level, there was no significant difference in alcohol+lead groups (P>0.05). Compared with lead groups at the same dose level, there was also no significant difference in alcohol+lead groups (P>0.05). There was an antagonistic interaction between alcohol and lead on NSC Suvivin mRNA expression.Totally, alcohol combined with lead inhibit the self-renewal of neural stem cells and induce apoptosis.The main combined effects of alcohol and lead are synergistic interaction. |
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