| Part 1:Anatomy of inferior parathyroid gland vessels in New Zealand white rabbitsObjective:To dissect and describe the location of inferior parathyroid glands and vascular characteristics of New Zealand white rabbits.Methods:The inferior parathyroid gland and its blood vessels in New Zealand white rabbits were explored.Results:1.The location of parathyroid gland in New Zealand white rabbits:type A is compact type,type B is isolated type.Among them,type B is divided into three types:type B1(parathyroid is above the upper pole of thyroid);type B2(parathyroid is at the horizontal area of thyroid);type B3(parathyroid is at the lower pole of thyroid below the horizontal area).According to the location of inferior parathyroid glands,they can be divided into 5 types:type BⅠ(parathyroid gland is located in the tracheoesophageal groove);type BⅡ(parathyroid gland is located between sternum thyroid muscle and hyoid muscle of scapula);Type BⅢ(parathyroid located around the carotid sheath);type BⅣ(parathyroid located in the thymus area behind the sternum);type BV(parathyroid located in other locations).2.According to the number of blood vessels visible to the naked eye,the hypoparathyroidism of New Zealand white rabbits can be divided into 6 types:1+X blood vessels;2+ X blood vessels;2+X blood vessels;3+ X blood vessels;4 and more blood vessels.Conclusion:This paper puts forward the classification of inferior parathyroid gland locations and blood vessels in New Zealand white rabbits for the first time,which provides the anatomical basis for the future research of animal experimental models related to parathyroid in New Zealand white rabbits.Part 2:Verification of the theory that "secondary injury" of inferior parathyroid gland can be induced by secondary hypocalcemia caused bypostoperative parathyroid ischemia and calcium supplementation can inhibit the"secondary injury"Objective:To verify the theory that "secondary injury" of inferior parathyroid gland can be induced by secondary hypocalcemia caused by low calcium after operation of ischemic parathyroid,and to confirm that calcium supplementation can inhibit its" secondary injury".Methods:The "Hypoxia and hypocalcemia injury" model of parathyroid cells in vitro was established,cell proliferation was measured by CCK-8,cell apoptosis was measured by flow cytometry,PTH was measured by ELISA.These methods can be used to screen the volume fraction of O2 with damage effect.The experimented cells were divided into Control Group,Hypoxia+Low Calcium Group,Hypoxia Group and Hypoxia+Calcium Supplement Group.Cell proliferation was measured by CCK-8,cell apoptosis was measured by flow cytometry,PTH was measured by ELISA,Active-Caspase-3 and p38 MAPK expression was measured by WB.The animal model of parathyroid vascular injury in vivo was established and divided intoSham Operation Group,Model Group and Calcium Supplement Group.PTH was measured by ELISA after treatment on day 1,day 3 and day 7.Ca2+was measured by colorimetry.Tissue and cell morphology were observed by HE staining.Apoptosis was measured by TUNEL staining.Active-Caspase-3 and p3 8 MAPK expression was measured by WB.Results:1 Cell model of parathyroid gland in vitro:1.1 Hypoxia brings "primary injury" to parathyroid cells.1.1.1 The results of CCK-8 showed that the survival rate of parathyroid cells decreased with the decrease of O2 volume fraction.1.1.2 The results of flow cytometry showed that the apoptosis rate of parathyroid cells increased with the decrease of O2 volume fraction.1.1.3 The results of ELISA showed that PTH secretion of parathyroid cells decreased with the decrease of O2 volume fraction.1.2 Low calcium causes "secondary injury" to parathyroid cells injured by hypoxia.1.2.1 The results of CCK-8 showed that the survival rate of cells in Hypoxia+Low Calcium Group was significantly lower than that in Hypoxia Group.1.2.2 The results of flow cytometry showed that the apoptosis rate of cells in Hypoxia+Low Calcium Group was significantly higher than that of cells in Hypoxia Group.1.2.3 The results of ELISA showed that the level of PTH secretion in Hypoxia+Low Calcium Group was significantly lower than that in Hypoxia Group.1.2.4 WB results showed that the expression levels of Active-Casepase-3 and p38 MAPK in Hypoxia+Low Calcium Group were significantly higher than that in Hypoxia Group.1.3 Calcium supplementation can inhibit the "secondary injury" of parathyroid cells.1.3.1 The results of CCK-8 showed that the survival rate of cells in Hypoxia+Calcium Supplement Group was significantly higher than that in Hypoxia+Low Calcium Group.1.3.2 The results of flow cytometry showed that the apoptosis rate of cells in Hypoxia+Calcium Supplement Group was significantly lower than that in Hypoxia+Low Calcium Group.1.3.3 The results of ELISA showed that the level of PTH secretion in Hypoxia+Calcium Supplement Group was significantly higher than that in Hypoxia+Low Calcium Group.1.3.4 WB results showed that the expression levels of Active-Casepase-3 and p38 MAPK in Hypoxia+Calcium Supplement Group were significantly lower than that in Hypoxia+Low Calcium Group.2 Animal model of parathyroid gland in vivo:Low calcium can bring "secondary injury" to parathyroid cells with ischemic injury,and calcium supplementation can inhibit "secondary injury" of parathyroid cells.2.1 The results of colorimetry showed that the level of serum Ca2+after treatment was the same before treatment.2.2 The results of ELISA showed that the PTH of the Model Group was significantly higher at the first day and the third day than before treatment.2.3 The results of HE staining showed that fibrosis of parathyroid tissue could be seen at the first day,the area of fibrosis increased at the third day,and it became more obvious at the seventh day.2.4 The results of TUNEL staining showed that calcium supplementation could reduce the apoptosis rate at the first day and the third day.2.5 The results of WB showed that calcium supplementation could reduce the expression of p38 MAPK and Active-Caspase-3.Conclusion:This study established the "ischemia and low calcium injury" model,in vitro "hypoxia and low calcium injury" model,in vitro and in vivo "injury calcium supplement" model of New Zealand white rabbit parathyroids.And it confirmed that low calcium can bring "secondary injury" to parathyroid cells injured by hypoxia,and aggravate the apoptosis of parathyroid cells,p38 MAPK participated in this process,and calcium supplementation can inhibit "secondary injury".Part 3:Mechanism of calcium supplementation on inhibiting apoptosis of parathyroid cells in "secondary injury"Objective:To explore the biological mechanism of calcium supplement inhibiting parathyroid "secondary injury" by inhibiting p38 MAPK protein expression.Methods:In order to optimize the conditions of parathyroid cell "secondary injury" in vitro cell model and injury-calcium supplement model,CCK-8 was used to measure the cell survival rate under different Ca2+ concentration conditions.Ca2+concentration with both significant inhibition and promotion of cell survival and the time point when it began to appear was selected.The cell model of parathyroid "secondary injury" in vitro was established.And the tested cells were divided into Control Group,Model Group,Calcium Supplement Group,p38 MAPK inhibitor Group,Calcium Supplement+p38 MAPK inhibitor Group,Calcium Supplement+ p38 MAPK agonist Group.PTH was measured by ELISA.Apoptosis was measured by TUNEL staining.The expression of Active-Caspase-3,Caspase-3,Caspase-6,Caspase-9,BCL2/BAX was measured by WB.p53,MAPKAPK2 and MEF2C was measured by QPCR.MMP was measured by JC-1.Results:1 To establish and optimize the cell model of "secondary injury" of parathyroid cells in vitro.1.1 The results of CCK-8 showed that the survival rate of parathyroid cells was significantly reduced after 12 h when the volume fraction of O2 was set to 1%and the concentration of Ca2+was 100 umol/L;when the final concentration of Ca2+was 500 umol/L,the survival rate of parathyroid cells in "secondary injury" could be improved,and the effect of promoting survival could be detected after 12 h.2 The biological mechanism of calcium supplementation inhibiting parathyroid"secondary injury" by inhibiting p38 MAPK protein expression.2.1 The results of PTH measured by ELISA showed that calcium supplement and p38 MAPK inhibitor could promote the recovery of PTH secretion in "secondary injury"cells.2.2 TUNEL results showed that calcium supplement and p38 MAPK inhibitor could reduce the apoptosis rate of those "secondary injury" cells.2.3 WB results showed that calcium supplement and p38 MAPK inhibitor could inhibit the expression of Caspase-6,Caspase-9,Active-Caspase-3..When increasing the expression of BCL2,decreasing the expression of Bax,and maintaining the expression of BCL2/BAX,no significant effect on the expression of Caspase-3 could be found.2.4 The results of QPCR showed that both calcium supplement and p38 MAPK inhibitor could decrease the transcription of p53 in "secondary injury" cells.There was no significant effect on MAPKAPK2 and MEF2C.2.5 The results of MMP and electron microscopy showed that both calcium supplement and p38 MAPK inhibitor could stabilize MMP in "secondary injury" cells and protect mitochondria.Conclusions:In this study,the conditions for optimizing the cell model of "secondary injury" and "injury calcium supplement" of parathyroid gland were established.It was confirmed that calcium supplementation could inhibit the apoptosis of parathyroid cells through p38 MAPK/p53 pathway,and inhibit the decrease of MMP by p38 MAPK expression. |
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