The Research Of Rat DRG Neuron Purification Via Differential Attachment And Complex Influence Of β3-tubulin Positive Axon From Corresponding Purification Culture Factors In DRG Tissue Culture

Objective: 1. To find out a simple method to purify the neurons; 2. To research the complex influence onβ3-tubulin positive axon by corresponding purification culture factors in Dorsal root ganglion (DRG) tissue culture. Methods : 1.⑴The DRGs were obtained from the neonatal SD microscopically, then, the DRGs were made into cell suspension via collagenase digestion and centrifuge;⑵The amount of attached DRG neurons and non-neuronal cells of three differential attachment groups, PDL group, PDL-LN group and Collagen-I group, in 30min, 60min and 100min was observed; 1.⑴The DRGs were obtained from the neonatal SD microscopically, then, the DRGs were prepared for tissue culture via collagenase digestion;⑵The extension and arborization ofβ3-tubulin positive axons was observed after 72h completely randomised DRG tissue culture for the research of the influences among culture substrates, serum and mitotic inhibitors. Results: 1.⑴The average amount of attached neurons in 30min in PDL group, PDL-LN group and Collagen-I group was 18, 25 and 5 respectively; The average amount of attached non-neuronal cells in 30min in PDL group, PDL-LN group and Collagen-I group was 34, 31 and 11 respectively; the amount difference between DRG neurons and non-neuronal cells of PDL group in 30min was statistically significant(P<0.05).⑵The average amount of attached neurons in 60min in PDL group, PDL-LN group and Collagen-I group was 58, 89 and 55 respectively; The average amount of attached non-neuronal cells in 30min in PDL group, PDL-LN group and Collagen-I group was 40, 37 and 40 respectively; the amount difference between DRG neurons and non-neuronal cells of PDL-LN group in 30min was statistically significant(P<0.05), however, this difference was composed of higher attached DRG neuron number and lower non-neuronal cell number.⑶The average amount of attached neurons in 100min in PDL group, PDL-LN group and Collagen-I group was 236, 360 and 136 respectively; The average amount of attached non-neuronal cells in 100min in PDL group, PDL-LN group and Collagen-I group was 107, 60 and 77 respectively; the DRG neurons and non-neuronal cells amount differences of all three groups in 30min were statistically significant(P<0.05), however, all these differences were composed of higher attached DRG neuron number and lower non-neuronal cell number. 2. The result of 72h DRG tissue culture:⑴Within the substrates factors, the best result ofβ3-tubulin positive axon extension and arborization was obtained when the substrate level is PDL-LN, the average number ofβ3-tubulin positive axon distal end was 0.243 ends per micron, and the average length ofβ3-tubulin positive axon was 1626.40±594.17μm, and the average length of PDL-LN level was statistically longer than the other two substrates, PDL and Collagen-I(P<0.05);⑵Within the serum factors, the highest number ofβ3-tubulin positive axon distal end was obtained in 5% concentration level, which is 0.25 ends per micron, but there was no statisticβ3-tubulin positive axon length difference to be observed among 0%, 5% and 10% levels(P>0.05);⑶Within the 5-FU factors, both the highest number ofβ3-tubulin positive axon distal end and the longestβ3-tubulin positive axon average length were obtained in 0μmol/L concentration level, which is 0.253 ends per micron and 1612.02±603.21μm respectively, and the difference between this 0μmol/L level and 20μmol/L,40μmol/L levels was static significant(P<0.05);⑷Within the Ara-C factors, the 0μmol/L level and 10μmol/L level got the same result ofβ3-tubulin positive axon distal end, which is 0.192 ends per micron, and this result was higher than 0.127 ends per micron which is the result of 20μmol/L level. And the longestβ3-tubulin positive axon average length was observed in 10μmol/L level, which is 1612.02±603.21μm long, and this result was statically higher than the other two levels(P<0.05). Conclusion: 1. A purified DRG neuron suspension for neuron culture could be obtained via PDL differential attachment for 30 min. 2. The DRG neuron culture mode, which composed of neuron special medium, PDL-LN substrate and 10μmol/L Ara-C, is recommended inβ3-tubulin positive axon research.