Purification of natural antikeratin autoantibodies from normal human serum and their effect on human keratinocytes cultured in vitro.

BACKGROUND: Antikeratin (AK) autoantibodies, circulating antibodies against epidermal keratins, have been detected in all normal human sera. However, direct evidence on the biological significance of AK autoantibodies is still lacking. OBJECTIVES: To purify AK autoantibodies from human serum and to make a preliminary study of their biological effects on human keratinocytes. METHODS: We first extracted keratin polypeptides from human stratum corneum and analysed their purity using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Next, a keratin affinity column was prepared with the extracted keratins, and AK autoantibodies were purified from pooled normal human serum. Antibodies obtained were identified with SDS-PAGE, enzyme-linked immunosorbent assay, immunoperoxidase staining, immunoelectron microscopy and Western blotting. The biological effect of AK autoantibodies on cultured human keratinocytes was studied using a DNA synthesis assay, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric determination and cell cycle analysis. RESULTS: On average, 1.83 +/- 0.24 mg of antibodies could be purified from 10 mL of pooled human serum. High-titre IgG (about 1 : 70) and low-titre IgM (about 1 : 30) AK autoantibodies were obtained. The DNA synthesis assay and MTT colorimetric determination demonstrated that AK autoantibodies have a significant dose-dependent inhibitory effect on cultured keratinocytes. Correlation coefficients in the two experiments were - 0.583 and - 0.797, respectively. Cell cycle analysis indicated that a small dose of AK autoantibodies leads to inhibition of proliferation of cultured keratinocytes, whereas a large dose of AK autoantibodies causes a visible hypodiploid peak, suggesting apoptosis of keratinocytes. CONCLUSIONS: The present research lays a solid foundation for further investigation into the biological significance of natural AK autoantibodies.

[The influence of acetylcholine on the proliferation of cultured human pituitary tumour cells].

AIM: To study the influence of acetylcholine on the proliferation, DNA synthesis and cell cycle of cultured human pituitary tumour cells. METHODS: MTT method, 3H-TdR incorporation and cell cycle analysis were used to examine the changes of proliferation and DNA synthesis of human pituitary tumour cells. RESULTS: Ach at 10(-7) mol/L - 10(-5) mol/L could decrease the 3H-TdR incorporation and the MTT A value in a dose dependent manner (P < 0.01), and the ratio of G1 phase of pituitary tumour cells increased markedly (P < 0.01). The effect of acetylcholine on the proliferation of cultured human pituitary tumour cells could be inhibited by atropine. CONCLUSION: Ach inhibited the proliferation, DNA synthesis and cell cycle of cultured human pituitary tumour cells, and the inhibitory effect was mediated by acetylcholine receptor.

[Experimental studies of genistein on the proliferation and apoptosis of human prolactinoma cells cultured in vitro].

AIM: To study the influence of genistein (GST) on the proliferation and apoptosis of cultured human prolactinoma cells. METHODS: MTT method and 3H-TdR incorporation and cell cycle analysis were used to examine the changes of proliferation and DNA synthesis of human prolactinoma cells under influence of GST and beta-estradiol (E2). Tdt-mediated dUTP nick end labeling (TUNEL) were employed to observe the effect of GST and E2 on the apoptosis of human prolactinoma cells. RESULTS: In a dose dependent manner, GST of different concentration could significantly inhibit the proliferation of human prolactinoma cells cultured in vitro. GST(10(-5) mol/L) could increase the proportion of cells in G1 phase from 55.3% up to 90.3%. E2 of different concentration could dose-dependently increase the proliferation of human prolactinoma cells. E (10(-5) mol/L) could increase the proportion of cells in G2 phase from 15.6% up to 41.8%. However, a lower suppressive proliferation of cultured human prolactinoma cells was observed with GST and E2 together. GST, not E2, could significantly induce the apoptosis of human prolactinoma cells cultured in vitro. CONCLUSION: GST inhibits the proliferation, DNA synthesis and cell cycle of cultured human pituitary cells, and induces its apoptosis. E2 decreases partly the effect of GST on the suppression of proliferation, not apoptotic induction, of human prolactinoma cells cultured in vitro.