| Background: According to recent statistics of Ministry of Health, about 16 million babies had been born each year in China and the rate of birth defect is about 4-6%(that is, millions of babies who were birth defect per year), but these data only shows visible deformity of birth defect. In addition, fetal who are in the adverse intrauterine environment and influence from abnormal factors of matrix, the normal development of the embryo in the whole, organ, cell and molecular level may have a series of changes, these changes can not cause "visibility" deformity in anatomical way, but will also set a dangerous precedent for individual health problems after birth. This is "FOAD"(fetal origins of adult diseases) in the field of medical research at present.FOAD which refers to the adverse intrauterine environment and abnormal maternal factors(e.g., high sugar, high salt, poor nutrition, hormone, etc.) in early embryonic development, the fetus, such as an early stage of human development, lead to the corresponding changes for the early development of early child in the cell and molecular level, and then to the structure and function for individual organs and overall system have long-term influence, makes the fetus have a higher prevalence to certain diseases(hypertension, diabetes, cancer, mental illness, etc.) during adults period. A prototype of this concept, as seen in David Barker, a professor in 1989 as a landmark epidemiological studies, show that there is a certain positive correlation between low birth weight and adult cardiovascular disease mortality, followed a large amount of literature to support the development of cardiovascular disease origin hypothesis. "The Fetal Origins Hypothesis" is also the "imprinting" effect theory. Nearly 60 years, animal experiments and clinical studies have found new evidence to support and develop the theory. Such as high sugar diet and high salt diet during pregnancy will have irreversible influence for RAS system of adult offspring, involving the multiple system and multiple organs. Recent research in this laboratory shows that maternal during pregnancy exposure to nicotine can cause RAS system changes of adult offspring, reflect on the changes of multiple systems and multiple targets(AT1 and AT2 receptors in the expression of tissues and organs such as heart, kidney, brain, in the circulation RAS substrate, key enzymes and products).Infant Birth Weight less than 2500 grams are known as Low Birth Weight, usually by Preterm Birth(PB) or Intrauterine Growth Retardation(IUGR). According to statistics, the incidence of LBW is about 7-15%, is one of the important cause of perinatal death, LBW individuals in childhood and adulthood significantly increased the rates of morbidity and mortality. Therefore, further understanding and discussing the causes and pathogenesis of these diseases is very important. Sufficient oxygen supply is the necessary element of fetal growth and development, and a variety of physiological factors or pathological factors of the mother during pregnancy, such as gestational hypertension disease, anemia, heart failure, preeclampsia, renal failure, chronic nephritis, gestational diabetes, chronic obstructive pulmonary disease, smoking, lung disease, and diseases such as placenta and umbilical cord factors and adverse factors in the environment can reduce uteroplacental blood flow, lead to fetal hypoxia, affect the normal fetal growth and development, resulting in IUGR and LBW.The body’s immune system can maintain the normal immune function, to complete the body’s immune defenses, the immune stability and immune surveillance plays an important role, once immune dysfunction illness will happen, and even malignant tumor. Thymus is the important central immune organs, are the sites of T lymphocyte development and maturity. The number or functional abnormalities of T lymphocyte will cause the imbalance of the body’s immune status and influence the occurrence and development of a variety of diseases such as autoimmune diseases, chronic infectious disease and malignant tumor. For a long time, people think that genetic and environmental factors play an important role in the incidence of malignant tumor and autoimmune disease. In recent years, a large number of epidemiological studies have shown that poor intrauterine environment lead to fetal development "procedural mechanism", lead to the increased risk for malignant tumor and autoimmune disease. Interleukin 2(IL-2) known as T cell growth factor, because of promoting lymphocyte immune response in vitro, clinically used to strengthen T cell immune of the AIDS patients or patients with malignant tumor, or by blocking IL-2 and its receptors to suppress T cell immune response of graft. HIF-1α/IKKβ/NF-κB and HIF-1α/MEK1/2/ERK1/2 are two important signaling pathways which lead to decrease of IL-2 under hypoxia conditions. IL-2 and its receptors are involved in the thymus developmental disorders induced by hypoxia during pregnancy and key protein changes of the signal pathway in hypoxia condition is also not clear. At present, the "imprinting" effect on the fetus intrauterine hypoxia have some research on abroad, but the influence which lack of oxygen to the fetus during pregnancy for thymus development of the offspring and its mechanism research has not yet been reported.This research proposed pregnant BALB/c mice from GD4 to GD19 have been induced by hypoxia, IUGR mice model was established, and abdominal incision when GD19 and IUGR individuals can be calculated by measuring the fetal mice morphology index. With the normal group as control, using HE staining method in optical microscope pathological changes of thymus tissue structure of the hypoxia group are seen. By flow cytometry the changes of T lymphocyte subsets in the fetal and offspring thymus which are induced by hypoxia during pregnancy are detected. Then Real-time PCR and flow cytometry analyse cell apoptosis in thymus tissue; By ELISA we detect the changes of cytokines related to the thymus development; By Real-time PCR and western blotting we detect the expression of key proteins in the IL-2 signal pathway. In addition, with FTOC method, we add exogenous IL-2 in hypoxia group, and the development of the thymus is detected in order to verify the above conclusion. From the whole, tissues, organs, cell and molecular level, we study the effect of hypoxia on the development of offspring thymus during pregnancy, and preliminary discuss on the mechanism of thymus developmental disorders of offspring which are induced by chronic hypoxia during pregnancy.Part one: The influence of hypoxia during pregnancy on the morphology and thymus development and function of fetal and offspringObjective: To detect the influence of hypoxia during pregnancy on the morphology and thymus development and function of fetal and offspring. Methods: Pregnant BALB/c mice were exposed to hypoxia or normoxia during pregnancy and the thymuses of the fetal and offspring were tested. Results: Compared with the control group, hypoxia during pregnancy had transient influence on pregnancy mice diet, can significantly reduce the weight of fetal body, heart, liver, kidney, and lung, and significantly reduce the level of p O2, SO2% in the fetal blood. Chronic prenatal hypoxia significantly decreased fetal body weight(P<0.05). There was no significant difference in fetal thymus weight between the control and the hypoxic group(P>0.05). The ratio of thymus-to-body weight in the hypoxia fetuses was significantly increased(P<0.05). Histological analysis showed a smaller cortical zone in the thymus of the offspring exposed to hypoxia, with decreased T lymphocytes, indicating poor development of the thymus. Compared with the control group, there was a significant increase in percentage of DP T cells during the T cell developmental process, while percentage of SP T cells showed a trend of decline. Meanwhile SP T cells showed a concomitant and parallel trend in the spleen and blood. Conclusion: Prenatal hypoxia induced the developmental disorders of the thymus in fetal and offspring of BALB/c mice.Part two: The mechanisms of prenatal hypoxia on thymus developmental disorders in BALB/c mouse offspring.Objective: To investigate the mechanisms of prenatal hypoxia on thymus development in BALB/c mouse offspring in vivo. Methods: Prenatal BALB/c mice were randomly divided into the control and hypoxia group. Offspring at GD7 were dissected under anesthesia at the end of treatments. Contents of IL-1, IL-2, IL-4, IL-7, and IL-10 in thymus tissue of the offspring were measured with enzyme-linked immunosorbent assay(ELISA). The apoptosis rate of the thymus in offspring was detected by Flow Cytometry. The m RNA and protein expression of Caspase-3, Ki-67, HIF-1α, IKKβ, NF-κB, MEK1/2, ERK1/2 were detected by Real-time PCR and western blotting. Results: The expression of IL-2 was significantly reduced in thymus tissue of the PH offspring at 7 days old(P<0.05), while IL-4 and IL-7 were increased compared with the control(P<0.05). IL-1 and IL-10 were not significantly different in thymus tissue between the control and PH group(P>0.05). Prenatal hypoxia increased m RNA levels of Caspase-3 and decreased the expression of Ki-67. The apoptosis rate of thymus cells was up-regulated by prenatal hypoxia. There was an increase in thymus HIF-1α and IKKβ, and a decrease in phosphorylated NF-κB, MEK1/2, and phosphorylated ERK1/2 compared with the control. Conclusion: Chronic hypoxia during pregnancy induced thymus developmental disorders in BALB/c mouse offspring via down-regulation of IL2.Part three: The mechanisms of prenatal hypoxia on thymus developmental disorders in BALB/c mouse offspring in vitro.Objective: To investigate the effect of IL-2 signal pathway on thymus developmental disorders in BALB/c mouse offspring in vitro. Methods: Freshly isolated thymic lobes were used to establish FTOCs. The cultures were grown in the presence or absent of recombinant murine IL2 under hypoxia or normoxia condition. After 48 h in culture, thymocytes were isolated and analyzed using flow cytometry. The m RNA expression levels of IL2R-α(CD25), IL2R-β(CD122), and IL2R-γ(CD132) were detected by Real-time PCR. Results: By flow cytometry the detection showed that adding exogenous IL-2 in the hypoxia group compared to the control group, DP(CD4+CD8+) T cells had increased obviously(P<0.05), and the percentage of SP(CD4+CD8- and CD4-CD8+) T cells had decreased significantly(P<0.05); Compared with the control group, the expression of IL-2β(CD122) and IL-2γ(CD132) decreased significantly in hypoxia group(P<0.05). Conclusion: Adding exogenous IL-2 can not reverse the developmental disorders of T lymphocytes in the thymus, and this process is irreversible; IL-2 receptors may be damaged by hypoxia during pregnancy, and cause the thymus developmental disorders of the offspring. |
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