Objective To establish a primary culture of human melanocytes from tiny skin sheets harvested by using a suction blister method, to carry out a serial subcultivation of the melanocytes with human amniotic membrane (AM) as a scaffold, and to observe the influence of AM on the adhesion, proliferation and dendrite development of melanocytes. Methods Tiny skin sheets were collected from the flexual forearm or lower abdomen of a healthy male volunteer by a suction blister method and melanocytes in the skin sheet were counted following Dopa staining under a microscope. The trypsinized skin sheets were scraped with a scalpel to harvest melanocytes which were subjected to a primary culture. Then, the melanocytes were inoculated onto fresh or cryopreserved AM followed by a culture for various durations (4, 8 and 12 days). The morphology and dendrite development of melanocytes were visualized under an inverted microscope after dopa-staining, cell viability evaluated by MTT assay, the adhesion to AM examined by hematoxylin and eosin (HE) staining protocol. Results The density of melanocytes was 1543.1±13.3 cells per mm2 and 857.4±101.7 cells per mm2 in skin sheets obtained from the forearm flexure and lower abdomen of the volunteer, respectively. A skin sheet of about 25.1 mm2 from approximately two blister roof was required to ensure the success of primary culture of melanocytes within 1 month. After culture on fresh or cryopreserved AM for 4, 8, and 12 days, most melanocytes were bi-polar with extended slender dendrites compared with those cultured in common cell culture medium. HE staining showed that melancytes adhered and were evenly distributed on the basement membrane of AM. MTT assay showed that the AM inhibited the proliferation of melanocytes, and no statistical difference was observed in the inhibitory effect between fresh AM and cryopreserved AM (P＞ 0.05). Conclusions Enriched with melanocyes, flexural forearm is a preferable donor site to offer skin sheets for primary culture of melanocytes. Human AM could improve the adhesive growth and dendrite development of melanocytes, and may serve as a promising bioscaffold for in vitro expansion of melanocytes.

Objective:To analyze the normal expression levels of different lung cancer tumor markers(TM)in the cerebrospinal fluid and to explore the influence of serum TM levels and brain parenchymal metastasis,to more accurately determine whether the cerebrospinal fluid TM levels of patients with suspected meningeal metastasis is elevated.Methods:The clinical data of 80 patients diagnosed with non-lepto-meningeal metastasis at Tianjin Medical University Cancer Hospital between January 2015 and February 2024 were collected,including 16 patients without lung cancer and 64 patients with lung cancer.Normal TM levels in the cerebrospinal fluid of patients without lung cancer and the difference in TM levels between the cerebrospinal fluid and serum samples were analyzed.The correlation between serum and cerebrospinal fluid TM levels was also analyzed.We then compared the differences in TM levels in the cerebrospinal fluid between groups with brain parenchymal metastasis and without brain parenchymal metastasis.Results:Normal levels of TPSA,CA19-9,CEA,Cyfra21-1,and SCC in the cerebrospinal fluid were lower than those in the serum(P<0.05);however,the levels of ProGRP and NSE in the cerebrospinal fluid were higher than those in the serum(P<0.05).The levels of TPSA,SCC,ProGRP,NSE,CEA,CA19-9,and Cyfra21-1 in the cerebrospinal fluid did not correlate with those in the serum(all P>0.05).The cerebrospinal fluid levels of TPSA,SCC,ProGRP,and CA19-9 were not significantly increased in patients with brain parenchymal metastasis compared to those in patients without brain parenchymal metastasis(P>0.05).Al-though CEA and Cyfra21-1 levels increased(P<0.05),their median values increased by less than 2 times and were all within the reference range;whereas,the level of NSE in the group with brain parenchymal metastasis was lower than that in the control group.Conclusions:The basal levels of ProGRP and NSE in normal cerebrospinal fluid were significantly higher than those in the serum;whereas,the expression levels of other TM in the cerebrospinal fluid were significantly lower than those in the serum.Whether the levels of TM in the serum were elevated and whether brain parenchymal metastasis was present,did not have a clinically significant impact on the TM levels in the cerebrospinal fluid.