| 摘要: | Background: There is an increasing interest in the clinical applications of various human platelet lysates (HPLs) in regenerative medicine. The scientific rationale supporting the clinical benefits of these platelet preparations is thought to be related to their content in numerous growth factors. In particular, a dedicated purified HPL, rich of neurotrophic factors, has been shown to provide neuroprotection to dopaminergic neurons in Parkinson's disease (PD) models. Our preliminary studies are evidencing that HPLs contain extra-cellular vesicles including microvesicles (MVs), raising still unanswered questions on the potential contribution of these MVs to the functional activity of HPLs.
Aims: to assess (a) the functional activity of various HPLs intended for specific clinical indications (b) the content of MVs: the number, the size, the biological functions and their role in the neuroprotective activity of HPL.
Material and Methods: Human platelet concentrates (PCs) obtained from Taipei Blood Center (Guandu, Taiwan) were aliquoted and processed into different platelet lysates: PPL (Platelet Pellet Lysate); HPPL (Heat-treated Platelet Lysate); SCPL (Serum-converted platelet lysate); and HSCPL (Heat-treated Serum-Converted Platelet Lysate). The number and the size distribution of MVs were determined by dynamic light scattering (DLS), nanoparticle tracking analysis (NTA), tunable resistive pulse sensing (TRPS), transmission electron microscopy (TEM). MVs in HPLs were further isolated using size exclusion chromatography (SEC) column. MVs functional activity was assessed by capture assays designed to determine the expression of pro-thrombotic tissue factor (MP-TF) and phosphatidylserine (MP-PS). MVs surface marker expression was examined by western blot (WB) and a bioanalytical platform comprising biochips and analytical tools using surface plasma resonance and atomic force microscope (SPRi-AFM) (FEMTO-ST, France). HPLs were tested for their thrombin activity and anti-oxidative property. In addition, we initiated the assessment of the neuroprotective activity exerted by selected MVs preparations using an in vitro model of PD where LUHMES dopaminergic neurons are exposed to erastin neurotoxin.
Results: All HPL preparations contained an abundant number of MVs (1011~1012/mL). In DLS analysis, MVs in all HPLs exhibited a population size ranging from 40~300 nm. MVs could be successfully isolated from the HPLs by qEV Izon size exclusion chromatography and exhibited a population size distribution range of 40~260 nm; 50~200 nm; 120~270 nm and 150~220 nm for SCPL, HSCPL, PPL, and HPPL, respectively. The MV size was confirmed by NTA, TRPS, and TEM. None of the HPLs were found to have detectable functional TF-expressing MVs but significant differences in PS-expressing MVs could be found, consistent with the mode of preparation and concentration of the various HPLs. MVs in PPL and HPPL were detected expressing platelet markers CD41 and CD61, macrovesicle marker CD9 and exosome marker CD63. No thrombin activity was found in PPL and HPPL, but was detected in SCPL and HSPL. Antioxidative activity capacity in SCPL and HSCPL is significantly higher than in PPL and HPPL. MVs isolated from HPPL did not affect the LUHMES cell viability. When assessed at a dose of ~4x1010 to 2x1011 these MVs did not protect the LUHMES cells from erastin neurotoxicity.
Conclusions: All HPLs contain a high quantity of MVs. The size and number of MVs as well as functional activity differ among HPL preparations. The physiological implications, if any, of these differences, deserve further in vitro studies that will be conducted in the near future. |
