Effect Of Copper-loaded Silicate Nanoparticles On Proliferation, Differentiation And Transference Of Chick Intestinal Epithelial Cell

Aim: Chick intestinal epithelial cell (IEC) was primarily cultured in vitro, then IEC culture line was tried to establish. Effects of enzymes were compared on isolation and proliferation of IEC. Under the condition of the optimal enzyme, the optimal density of FCS, CO₂ and optimal temperature which prompted proliferation of IEC were confirmed. IEC culture line was used as a model to investigate the effect of Copper-loaded Silicate Nanoparticles (CSN) on the conformation, proliferation, differentation and migration of chick IEC.Methods: Various enzymes were employed for isolating the epithelial cells, the isolated cells were cultured in Dulbecco s Modified Eagle Medium containing 5% foetal calf serum at 39°C in a 5% CO₂ incubator . The cell growth and proliferation were observed by using some efficient methods such as MTT colorimetric assay and cell counting. The IEC state and conformation of growth were observed by inverted contrastaddition microscope and electronmicroscopy. IEC was identified by using histochemical staining phosphates and H-E staining.Results: The results showed that satisfactory isolation of the chick IEC and good attachment were achieved by using a combination of the 300U/mL crude collagenase XI and O.lmg/mL neutral protease . Intestinal mucus were digested with this combination for 40 minutes at 37℃, as a result many epithelial cells were collected by centrifugal effect. The epithelial cells were observed by invert contrast microscope, which were organoid units that remained as polarized intact layers, and the cells were globular or olivary, the cellular borderlines were very clear. Their cytoplasms were very rich and their cellular nuclei were round or scree-like in which the chromatin was very sparse, and there were one or two nucleolus in the nuclei.. A large number of viable IECs were cultured in DMEM containing 5% foetal calf serum at 39℃ in a 5% CO₂ incubator in vitro. After given time, IEC was characteristic of pre-crypt units by electronmicroscopy and confirmed the presence of microvilli of extracellular, with intercellular tight junction and desmosomes etc, intracellular mitochondria and endoplasmic reticulum etc. Cells attached for 1² days, proliferated rapidly for 6⁷ days, and reached confluence for 10-12 days. The cells grew in a monolayer arrangement and appeared as flat polygonal cells.Histochemical staining for alkaline phosphatase (ALP) revealed that ALP activity was expressed in (91.16±6.97)% attached cells on 7th day. These cells showed blue-black dyeing.The effect of CSN was comparatively studied by using cell culture. The result showed that the addition of 30μ g/mL or 50μg/mL CSN to DMEM did not bring about cytotoxin and change comformation of IEC as compared to the control. However, microvillis of cell were clearer than the free CSN in DMEM on the 10th day. Addition of CSN to culture promoted IEC proliferation and growth of IEC, which had completely reached confluence on 10th day, wherever control still had nonconfluent areas on 10th day. CSN could promote the number of migration of IEC per unit wounded area (P<0.05), suggesting that a positive action of CSN on intestinal mucosa may be to promote the rapid restitution ofthe damaged areas.Conclusions: The results showed that satisfactory isolation of the epithelia and good attachment were achieved by using a combination of the 300U/mL crude collagenase XI and O.lmg/mL neutral protease. This experiment also suggested that 2.5%⁵.0% FCS, 39°C, 5⁷.5%CO2 were most optimal conditions for the growth and proliferation of EEC. IEC were majority by microscopy and histochemical staining for alkaline phosphatase (ALP) research.Complement of30 u g/mLor 50 u g/mL CSN in DMEM promoted IEC proliferation .differentiation and the restitution of the damaged areas.