| Background Mesenchymal stem cells(MSCs)are a kind of stem cells,which have multiple potential of differentiation.On one hand,they have the capability of self-renew;on the other hand,they can differentiate into one or more kind(s)of cells under certain conditions.Recently,MSCs have been widely used in tissue engineering.In addition,they are also applied in the treatment of clinical diseases,such as bone disease and neurodegenerative disease.Although MSCs have good prospects for clinical application,the clinical translation is restricted because of its limited proliferation and differentiation in vitro.How to solve these problems has become a research hotspot and bottleneck in this field.In recent years,the effects of electromagnetic radiation on stem cells have been investigated.It is reported that exposure to electromagnetic radiation under some conditions could affect the proliferation and differentiation of MSCs.Till now,no literature is available concerning the biological effects of electromagnetic pulse(EMP)on MSCs.In addition,there are only a few reports about the biological effects of radio-frequency(RF)radiation on MSCs.Therefore,in this study,human umbilical cord mesenchymal stem cells(h UC-MSCs,one kind of widely used MSCs)were chosen to investigate the effects of EMP and 1950 MHz RF on the proliferation and differentiation in h UC-MSCs.Objective The objective of this study is to clarify the effects of EMP and 1950 MHz RF on the proliferation and differentiation in h UC-MSCs and elucidate the underlying mechanisms.Part ?: Primary culture and identification of h UC-MSCsMaterials and Methods1.The umbilical cords of newborns were collected from the hospital.After stripping the Wharton's jelly,tissue block method was used to culture h UC-MSCs.The cultured cells were digested by trypsin and sub-cultured when the cell density was about 80%.The third passage h UC-MSCs in logarithmic growth period were harvested and seeded into 96-well plates.The proliferation level of cells was detected by the CCK-8kit for 5 consecutive days and the growth curve was plotted.2.The protein levels of MSCs specific antigens such as CD90,CD105 and CD166 were detected by flow cytometry.Results1.It was found that some new cells appeared around the tissue block of Wharton's jelly after 7-day primary culture and the cell density reached over 90% after 14-day culture.The morphology of the cells was stable and had typical morphology of MSCs even after passage.The results from CCK-8 method showed that h UC-MSCs entered the logarithmic growth period on the second day after seeding and entered the platform stage on the fourth day after seeding.The doubling time of the cells was 32 h.2.The results of flow cytometry showed that the expression levels of CD90,CD105 and CD166 of the cells were high,and the percentage of positive cells was more than 96%.Part ?: Effects of EMP on the proliferation and osteogenic differentiation in h UC-MSCsMaterials and MethodsThe EMP signal was generated by SPG200-EMP equipment.The field strength was720 k V/m.The pulse width was 40 ns.The repetition frequency was 1 Hz.The number of pulses could be set from 1 to 6000.1.The effect of EMP on the proliferation in hUC-MSCs1.1 Cell grouping and EMP exposure: h UC-MSCs of the 3rd~6th passage in logarithmic phase were randomly divided into sham-exposure group and EMP exposure group which were exposed to EMP once or three times(once a day for consecutive 3 days)and the number of EMP pulses per time were 10,100 and 1000.1.2 Methods: The cell viability was detected by CCK-8 kit on the first day(1st),the third day(3rd),the 5th day and the 7th day after EMP exposure.The cell cycle distribution and apoptosis level were detected by flow cytometry.2.The effect of EMP on the osteogenic differentiation in h UC-MSCs2.1 Cell grouping and EMP exposure: h UC-MSCs of the 3rd passage in logarithmic phase were randomly divided into sham group,EMP group,OM(Osteogenic induction medium)group and OM+EMP group.The cells were sham exposed or exposed to EMP once,4 times and 7 times respectively(once a day)and the number of EMP pulses per time was 100 and 1000.2.2 Methods: The activity of alkaline phosphatase(ALP)in cells was determined by microplate method.The protein levels of osteogenic specific protein such as COL? and OPN were detected by western blotting(WB).The formation of calcified nodules was measured by Alizarin staining.Results1.The effect of EMP on the proliferation in h UC-MSCsThe results of CCK-8 kit showed that compared with sham group,the cell viability of h UC-MSCs in EMP group(exposure once,10 pulses)didn't change obviously on the 1st day,3rd day,5th day and 7th day after EMP exposure,the similar results were found in100 pulses group and 1000 pulses group on the 1st day after EMP exposure.However,the cell viability of h UC-MSCs decreased significantly on the 3rd day,5th day and 7th day in100 pulses group and 1000 pulses group after EMP exposure.There was no obvious difference in cell vitality between the 100 pulses group and 1000 pulses group.Compared with sham group,the cell viability of hUC-MSCs in EMP group(10pulses each time,once a day,for consecutive 3 days)didn't change obviously on the 1st day but decreased significantly on the 3rd day and 5th day after EMP exposure(P < 0.05 and P < 0.01,respectively).However,the cell viability reduced significantly on the 1st day,3rd day and 5th day in 100 pulses group and 1000 pulses group after EMP exposure.There was no significant difference in cell viability between the 100 pulses group and1000 pulses group.Compared with the cells exposed to 10 pulses EMP once,the cell viability of h UC-MSCs exposed to 10 pulses EMP for three times(once a day)didn't change obviously on the 1st day but decreased significantly on the 3rd day and 5th day after EMP exposure.The cell viability of h UC-MSCs exposed to 100 pulses and 1000 pulses EMP for three times(once a day)reduced dramatically on the 1st day,3rd day and 5th day after exposure compared with cells exposed to 100 pulses and 1000 pulses EMP one time respectively.The results of flow cytometry showed that compared with sham-group,the cell cycle distribution of h UC-MSCs significantly changed at 24 h after one time EMP exposure(100 pulses).For example,the proportion of cells in S phase decreased significantly(P <0.01),and the proportion of cells in G1 phase increased significantly(P < 0.05).The level of cell apoptosis didn't vary significantly after exposure to EMP.2.The effect of EMP on osteogenic differentiation in h UC-MSCsThe results of ALP activity detection showed that the ALP activity of h UC-MSCs did not change compared with sham group on the 21 th day after one time EMP exposure(100pulses and 1000 pulses).However,the ALP activity of cells in OM +EMP group increased markedly compared with OM group on the 21 th day of osteogenic induction under the same EMP exposure condition(P < 0.05).The ALP activity in h UC-MSCs did not change after exposing cells to EMP for 7times(100 pulses each time,once a day,for 7 days),compared with sham group.However,compared with OM group,the ALP activity in OM+EMP group increased remarkably on the 21 th day after exposure to 100 pulses EMP once(P < 0.05).After exposure to 100 p EMP exposure for 4 times,the ALP activity in OM+EMP group increased remarkably compared with OM group on the 14 th day and 21 th day of osteogenic induction(P < 0.05 and P < 0.01,respectively).The ALP activity in OM+EMP group(100 pulses EMP exposure for 7 times)increased remarkably on the 7th day,14 th day and 21 th day of osteogenic induction compared with OM group(P < 0.05,P < 0.05 and P < 0.01,respectively).After exposure to EMP for 7 times(once a day for consecutive 7 days),the protein expression of COL I and OPN in h UC-MSCs was enhanced significantly on the 14 th day and 21 th day of osteogenic induction compared with OM group(P < 0.01).The results of Alizarin red staining showed that the area of calcified nodules increased significantly on the 21 th day of osteogenic induction under the same EMP exposure condition(P < 0.01).Part ?: Effects of 1950 MHz RF on proliferation and osteogenic differentiation in h UC-MSCsMaterials and MethodsThe RF exposure frequency was 1950 MHz.The specific absorption rate(SAR)could be set from 0 to 4.0 W/kg.The exposure time was adjustable.1.The effect of RF on the proliferation in hUC-MSCs1.1 Cell grouping and RF exposure: h UC-MSCs of the 3rd~6th passage in logarithmic phase were randomly divided into sham group and RF group.The cells were sham exposed or exposed to 1950 MHz RF with different SAR value(0.5,1.0and 2.0 W/kg)for consecutive 7 days(1 h/d,5 min on/10 min off).1.2 Methods: The cell viability was detected by the CCK-8 kit for consecutive 6 days after RF exposure.Cell cycle distribution was detected by flow cytometry,and the protein level of proliferation related protein Ki67 was detected by immunofluorescence staining.2.The effect of RF on the osteogenic differentiation in h UC-MSCs2.1 Cell grouping and RF exposure: h UC-MSCs of the 3rd passage in logarithmic phase were randomly divided into sham group,RF group,OM group and OM+RF group.The cells were sham exposed or exposed to 1950 MHz RF with SAR value of2.0 W/kg for consecutive 7 days(1h/d,5 min on/10 min off).2.2 Methods: The ALP activity in cells was measured by the microplate method immediately after 7-day RF exposure.Results1.The results of CCK-8 kit showed that compared with sham group,the cell viability of h UC-MSCs did not change significantly after RF exposure for consecutive 7 days with different SAR values.Similarly,the results of flow cytometry and immunofluorescence showed that the cells cycle distribution and protein level of Ki67 did not change obviously after RF exposure.2.The results of ALP activity detection showed that compared with sham group the ALP activity of h UC-MSCs in RF group didn't change significantly after RF exposure for consecutive 7 days(SAR 2.0 W/kg),and compared with OM group,the ALP activity of h UC-MSCs in OM+RF group didn't change significantly.Conclusions1.10 pulses EMP exposure for one time with EMP field strength 720 k V/m,pulse width40 ns and repetition frequency 1 Hz could not affect the proliferation in h UC-MSCs,however,EMP exposure for 3 times could inhibit cell proliferation.When the EMP pulses increased to 100 and 1000,the inhibition effects on cell proliferation became much more obvious.EMP exposure for multiple times also has much more obvious effects on cell proliferation.EMP exposure could not cause apoptosis in h UC-MSCs.2.100 pulses and 1000 pulses EMP exposure alone(field strength 720 kV/m,pulse width 40 ns and repetition frequency 1 Hz)could not induce osteogenic differentiation in h UC-MSCs.However,it could enhance the effect of osteogenic induction medium-induced osteogenic differentiation in h UC-MSCs,and EMP exposure for multiple times had much more obvious effects.3.Exposure to 1950 MHz RF with different SAR values(0.5,1.0 and 2.0 W/kg)for consecutive 7 days could not affect the proliferation in h UC-MSCs.4.Exposure to 1950 MHz RF with SAR value 2.0 W/kg for consecutive 7 days could not induce osteogenic differentiation in h UC-MSCs,similarly,it could not affect osteogenic induction medium-induced osteogenic differentiation in h UC-MSCs. |
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