Effect Of Low Concentration Glucose On Human Intervertebral Disc Nucleus Pulposus Cell Morphology, Growth And Apoptosis In Vitro

Among the factors that contribute to the intervertebral disc degeneration, the nutrient deprivation is the main one. Nutrition penetrates into the intervertebral disc via two routs: from the blood vessels surrounding the annulus and from the blood vessels at the cartilage end-plate. As the largest vascular tissue in the human body, the intervertebral disc is often in the risk of lack of nutrient supply. The cartilage endplate gradually becomes calcified and thin, and its blood vessel number keeps reducing with the growth of one's age. The nutrient deprivation in the intervertebral disc can lead to the increment of the apoptosis rate of nucleus pulposus cells, and further to disc degenerating. Glucose is one of nutritional elements for nucleus pulposus cells in the intervertebral disc. The energy for intervertebral disc cells is provided in the glycolytic pathway. Therefore, a large number of glucose is consumed in the intervertebral disc cells. However, the calcification of the cartilage end plate can limit the penetration of glucose into the nucleus pulposus, which results in the decrease of glucose concentration in the nucleus pulposus, the reduction of cellular vitality and the increasing rate of apoptosis. This experiment is to study the effect of low concentration glucose on the morphology,the growth and the apoptosis of nucleus pulposus cell so as to provide evidence for the role of glucose in the degeneration of the intervertebral disc.This study was divided into two parts. In the first part, nucleus polposus cells were cultured in vitro. The specimens were taken from patients with scoliosis in their orthopedic operations, who are selected according to their ages ranging from 10 to 15. The nucleus pulposus in each specimen was cut into pieces after all the ligament, annulus fibrosus and cartilage end plate being removed from the specimens. The nucleus pulposus were digested in the incubator for 40min, in which there was a mixture containing 0.25% trypsin, 0.2%Ⅰcollagenase and 0.2%Ⅱcollagenase. DF12 + 10% FBS was used to terminate the process of digestion after nucleus pulposus turned flocculent. The tissue residue centrifugation was removed and DF12 +15% FBS was added in the cultivated medium. Then, these cells were seeded in culture flasks and placed in the incubator. Growth curve of these cultured cells was draw by MTT;The changes of the cell morphology were observed by phase contrast microscope; Growth curve of these cultured cells was draw by MTT; The cell morphology was also observed by the toluidine blue staining; and the cell phenotype was identified by GAG staining and typeⅡcollagen immunehistochemical staining. The results showed as following, the cultured cells were nucleus pulposus cells by GAG staining and typeⅡcollagen immunohistochemical staining. The morphology of these cells was good by toluidine blue staining. The growth curve of these cultured cells displayed that cells grew well. It can be concluded that the morphology and the vitality of the cultured nucleus pulposus cells were well. The cell phenotypic expression was nucleus pulposus cell and these cells contented the need of experimental study in vitro.The second part is to investigate the influence of different glucose concentrations medium on the morphology, growth and apoptosis of nucleus pulposus cells in vitro. The nucleus pulposus cells, cultured in the first part of this experiment, were divided into four groups. The human nucleus pulposus cells were cultured in the four kinds of solutions with the control group (5mmol/L), Group 1(2.5mmol/L), Group 2(1mmol/L) and Group 3(0mmol/L). After cultured for 48h and 72h, cells morphological changes were observed by the phase contrast microscope. The ultra-structural changes of these cultured cells were observed by transmission electron microscope. The apoptosis rate was detected by flow cytometry. It was found that the number of cells increased gradually with the increment of glucose concentration. The cells in the control group, observed with the phase contrast microscope, were polygonal or in the shape of short spindle, rich in cytoplasm and in good growth. However, lots of cells in the shape of long spindle, short of cytoplasm and low refractivity, which showed the ageing of cells in 2.5mmol/L,1mmol/L,0mmol/L group. In 2.5mmol/L group and 1mmol/L group, a number of lysoomes and fat droplets were found in the nucleus pulposus cells by transmission electron microscope. In 0mmol/L group, the number of rough endoplasmic reticulum was decreased, organelles were damaged, cell membrane was not complete and some vacuoles and phagocytosis were found in some nucleus polposus cells. The apoptosis rates were detected by the flow cytometry showed that it decreased with the glucose concentration increasing. It can be concluded that the proliferation of the human nucleus polposus cells was inhibited and the apoptosis rate was increased with the decrease of glucose concentration. The morphology of nucleus polposus cells was changed at the same time.