Construction and application of differentially regulated tissue-engineered nerve grafts

Promoting Effects of Neuroligin-1 and Neuroligin-2 on Synapses in Motor and Sensory Neurons

[0038](1) Culture of motor neurons: a SD rat pregnant for 15 d-16 d was dissected under an anatomical lens to remove ventral tunica, dorsal root ganglia and blood vessels of a spinal cord, and a ventral spinal cord of a fetal rat was carefully separated and placed in a petri dish containing ice-cold anatomical fluid. A tissue piece having a size of 0.5 cm3 was cut by a microscissor and then transferred to a 15 mL centrifuge tube. After adding 0.25% trypsin and 200 U / ml DNase, digestion was performed at 37° C. for 30 min, and then digestion was terminated by adding DMEM+10% FBS. Centrifugation was performed at 1000 r / min for 5 min, and supernatant was discarded. 5 mL of Optiprep separation solution was added, and density gradient centrifugation was performed at 3000 r / min for 15 min. Then liquid in the centrifuge tube was divided into 3 layers, wherein the middle layer contained spinal motor

Promoting Effect of Neurexin-1β in Combination with Neuroligin-1 or Neuroligin-2 on Synapse Formation

[0043]Motor neurons and sensory neurons were cultured for 10 d in vitro and then divided into groups as follows. Neurexin-1β in combination with Neuroligin-1 or Neuroligin-2 was added to motor neuron and sensory neuron media, respectively:

[0044]Motor Neuron Group:

[0045](1) Neurexin-1β (final concentration of 200 ng / mL)+Neuroligin-1 (final concentration of 200 ng / mL);

[0046](2) Neurexin-1β (final concentration of 1 μg / mL)+Neuroligin-1 (final concentration of 200 ng / mL);

[0047](3) Neurexin-1β (final concentration of 1 μg / mL).

[0048]Sensory Neuron Group:

[0049](4) Neurexin-1β (final concentration of 200 ng / mL)+Neuroligin-2 (final concentration of 200 ng / mL);

[0050](5) Neurexin-1β (final concentration of 1 μg / mL)+Neuroligin-2 (final concentration of 200 ng / mL);

[0051](6) Neurexin-1β (final concentration of 1 μg / mL).

[0052]The culture was continued for 24 h. Results of immunocyt

Construction of a Differential Tissue-Engineered Nerve According to the Present Invention

[0055]1. Preparation of Sustained-Release Microspheres Containing Neuroligin-1, Neuroligin-2 and Neurexin-1β Recombinant Protein

[0056]Polylactic acid-glycolic acid copolymer (PLGA, a molar ratio of lactic acid to ethanol was 53:47, and molecular weight [MW] was 25 kDa). PLGA microspheres were prepared by a water / oil / water multiple emulsion method. 100 mg of PLGA was dissolved in 1 mL of dichloromethane, and then emulsified into 3 mL of a 7% (w / v) poly(vinyl alcohol) (PVA) aqueous solution, and ultrasonicated for 1 min to prepare an emulsion of a first step. Then, the above solution was added to 50 mL of a 1% (w / v) poly(vinyl alcohol) (PVA) aqueous solution (containing 2% isopropanol), and ultrasonicated again for 3 min to form an emulsion of a second step. The emulsion of the second step as mentioned above was slowly stirred overnight at room temperature, and the residual dichloromethane wa