human fibroblasts

When LDL-ferritin is cultured with regular human fibroblasts, surface-bound ferritin cores may be seen under an electron microscope. The ferritin cores that were coupled to the membrane's surface were concentrated there and covered with a fuzzy substance that matched covered areas on other cell types. Low-density lipoprotein was added to patient fibroblasts before being incubated. These patients have familial hypercholesterolemia in a homozygous form. Despite the transformed cells having an equal number of sunken coated membrane regions, LDL-ferritin bound to cell membranes was not identified (Anderson, Goldstein, & Brown, 1976).
It has been discovered that normal human fibroblast monolayers include particular receptor sites for LDL-ferritin and other low-density lipoproteins. The reason for this study was to locate low-density lipoprotein receptors which are absent on a familial hypercholesterolemia homozygous fibroblast but available on the plasma membrane of a normal human fibroblast. Patients with a receptor negative form of homozygous and other healthy subjects provided skin biopsy extracts which provided ten cultured samples of fibroblasts.

These were studied in a growth phase where forty-eight-hour incubation was done with the lipoprotein-deficient serum present. Human LDL and lipoprotein deficient serum were attained from the plasma of healthy patients and arranged using electro centrifugation. The ferritin was activated using the technique of Kishida separated from glutaraldehyde, then mixed with purified LDL in a ferritin. Monolayers in fibroblast were put in a cold room for 30 minutes, then removed, and placed with cold human lipoprotein deficient serum and LDL ferritin.

After the incubation, the monolayers were washed and buffered. Further fixation was done whereby the monolayer cells are dehydrated in ethanol solutions and then washed. From the monolayers cells, discs of fifteen millimeters’ diameter were punched out. Fresh Epon was then used to stick the discs together. Finally, sections of these monolayers were then made along a longitudinal plane. The longitudinal plane helps to determine the levels of LDL ferritin that is present on the cells’ surfaces. The cells were then scrutinized to show some ferritin cores on surfaces of the cells.

It was detected that LDL ferritin complex bound especially to confined regions on the plasma membranes of on normal fibroblast on both upper and lower parts. Using the method of indentation and localization, these areas were observed to be indented and coated with glycocalyx. Further analysis indicated that a small proportion was part of uncoated regions, found on the cell surface. Fibroblast from some mutant homozygote reared with LDL ferritin in the same settings as normal fibroblast showed no ferritin cores bound on their plasma membrane. The numbers of ferritin cores associated with fibroblasts plasma membrane were then quantitated. Afterward, incubation using the LDL ferritin was performed on the mutant homozygote cells and the normal cells. Binding agents were present during the incubation process, and their effects on the binding tested.

This study demonstrated that normal human fibroblast has receptors for LDL ferritin on their plasma membrane. LDL ferritin binding is not observed on the membranes of cells from a familial hypercholesterolemia homozygous which have revealed biochemically to contain some deficiencies in the LDL cell receptors. More so, a specific binding was observed on the plasma membrane. Also, there was an inconsistent level of LDL ferritin shown to abide by the proteinaceous layer which covers the surfaces of these cells. A same number of LDL ferritin can be found on both normal of familial hypercholesterolemia homozygote cells. This proteinaceous material is believed to represent the nonspecific sites for binding.

This research provides a thorough evidence of low-density lipoprotein receptors that is present in the membrane of normal fibroblast which is not present in cells of a familial hypercholesterolemia homozygote fibroblast. This research lays a basis of further follow-up studies to assess if the LDL receptors can move to the plasma membrane which contains the coated regions or if they are in specific areas.

The main point to learn from this research is that some low- density lipoprotein receptors may be present in the plasma membrane of a normal human fibroblast. Similarly, these receptors proteins may also be absent on a homozygous fibroblast. The researchers used the method discussed above as it provides clear and precise results in showing the location of the lipoprotein receptors. If other methods could be used, same results would be obtained. The study above clearly supports the objective of this research which was meant to show low-density lipoprotein receptors can be found on the plasma membrane but absent from a familial hypercholesterolemia homozygote. In future studies, researchers might out to use other methods to investigate the objective of this study rather than the method discussed above.



Reference

Anderson, R. G. W., Goldstein, J. L., & Brown, M.S. (1976). Localization of low-density lipoprotein receptors on the plasma membrane of normal human fibroblasts and their absence in cells from a familiar hypercholesterolemia homozygote. Cell Biology, 73(7), 2434-2438.