No abstract

Poisk optimal'nykh kletochnykh modeley dlya izucheniya patogeneza giperplasticheskikh rubtsov yavlyayetsya aktual'noy zadachey. Tsel'yu issledovaniya bylo otsenit' perspektivu ispol'zovaniya telomerizovannykh fibroblastov v kachestve ob"yekta pri 3D-modelirovanii patologicheskikh gipertroficheskikh rubtsov in vitro. Kletki NF i Fb-hTERT kul'tivirovali v vide monosloya i sferoidov, v intaktnom sostoyanii i pri vozdeystvii TGFb1. Metabolicheskuyu aktivnost' kletok otsenivali metodom MTT. Skorost' zarastaniya defekta monosloya kletok vychislyali s pomoshch'yu scratch-testa. Uroven' ekspressii genov, assotsiirovannykh s giperplasticheskimi protsessami, opredelyali metodom qRT-PCR. Dlya Fb-hTERT kharakterno boleye vyrazhennoye po sravneniyu s NF usileniye metabolicheskoy aktivnosti kletok pri vozdeystvii TGFb1 (dlya 1 ng/ml 179 ± 12% i 135 ± 13% sootvetstvenno; p < 0,05). Sferoidy, sformirovannyye iz Fb-hTERT, byli znachitel'no krupneye sferoidov, poluchennykh iz NF. Produktsiya osnovnykh spetsifichnykh dlya fibroblastov belkov, assotsiirovannykh s produktsiyey vnekletochnogo matriksa (COL1A1, COL3A1, FN1), pri stimulyatsii TGFb1 byla nizhe v Fb-hTERT po sravneniyu s NF (boleye chem v 25, 20 i 2 raza sootvetstvenno; p < 0,05). Intaktnyye NF boleye aktivno, po sravneniyu s Fb-hTERT, vosstanavlivali defekt monosloya (v 2,28 raza na vtoryye sutki; p < 0,05). Pri etom vozdeystviye TGFb1 privodilo k uvelicheniyu skorosti zapolneniya defekta kletkami Fb-hTERT (v 2 raza na vtoryye sutki; p < 0,05), no ne NF. Takim obrazom, telomerizovannyye fibroblasty imeyut ryad fenotipicheskikh priznakov, kharakternykh dlya keloidnykh fibroblastov, no v to zhe vremya yest' ogranicheniya, kotoryye sleduyet uchityvat' pri ispol'zovanii Fb-hTERT dlya modelirovaniya patologicheskikh gipertroficheskikh rubtsov.

The search for the optimal cell model for studying the pathogenesis of pathological scars is a pressing challenge. This study aimed at evaluating the feasibility of using telomerized fibroblasts for the in vitro 3D modeling of pathological hypertrophic scars. NF and Fb-hTERT cells were cultured as monolayers and spheroids in the absence and in the presence of TGFβ1. The metabolic activity of the cultured cells was assessed using the MTT assay. Cell migration was estimated using the scratch assay. The expression of genes associated with fibrous scar tissue growth was measured by qRT-PCR. Fb-hTERT cells were more metabolically active than NF cells in the presence of TGFβ1 (for 1 ng/ml: 179 ± 12% vs. 135 ± 13% respectively; p < 0,05). Spheroids grown from Fb-hTERT cells were significantly larger than those derived from NF cells. In the presence of TGFβ1, the expression of proteins associated with extracellular matrix production (COL1A1, COL3A1, FN1) was lower in Fb-hTERT cells than in NF cells (more than 25, 20 and 2-fold, respectively; p < 0.05). Intact NF cells were more active in closing the scratch than Fb-hTERT cells: on day 2, the gap closure rate was 2.28 times higher in NF cells (p < 0.05). Exposure to TGFβ1 stimulated Fb-hTERT, unlike NF cells, to close the gap 2 times faster on day 2 (p < 0.05). Thus, telomerized fibroblasts have a few phenotypic traits observed in keloid fibroblasts; still there are some limitations that should be accounted for when using Fb-hTERT cells for the modeling of pathological hypertrophic scars.